INFORMATION TO USERS
This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.
The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.
In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.
Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.
University Microfilms International A Beil & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 0227321
Effects of antimitotic agents on cultured adrenal chromaffin cells: Implications for microtubule involvement with adrenal nicotinic receptors
Lopez, Isabel, Ph.D.
The Ohio State University, 1992
UMI 300 N. Zeeb Rd. Ann Arbor, Ml 48106 EFFECTS OF ANTIMITOTIC AGENTS ON CULTURED ADRENAL
CHROMAFFIN CELLS: IMPLICATIONS FOR MICROTUBULE
INVOLVEMENT WITH ADRENAL NICOTINIC RECEPTORS
A Dissertation
Presented in Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of The Ohio State University
By
Isabel Lopez, B.S., M.S.
*****
The Ohio State University
1992
Dissertation Committee: Approved by
Dennis B. McKay, Ph.D.
Norman J. Uretsky, Ph.D.
Allan M. Burkman, Ph.D.
Lane J. Wallace, Ph.D. Dennis B. McKay, Ad College of Pharmacy To Tim and My Mom and Dad
ii ACKNOWLEDGEMENTS
I would like to extend my deepest and most sincere gratitude to Dr. Dennis B. McKay for his limitless patience, guidance and support throughout the course of my research, but most important for his friendship and encouragement which will never be forgotten.
I also would like to extend my appreciation to the entire faculty of the College of Pharmacy, Division of Pharmacology for their ready and willing guidance and insight throughout this study and the knowledge shared.
I would like to express my sincere thanks to all of the graduate students past and present that I have had the pleasure of becoming friends with both in and out of the laboratory. All have made this an enjoyable working environment. I also would like to thank Susan McKay, Jennifer Maurer and Patricia Trent-Sanchez for their scientific comments in the subject area and for their technical help throughout this research.
To my parents, Vicente and Vicki, thank you so much for your loving guidance, support and patience through the years and for your unending concern and support for my education even when it has meant time away from you.
Finally, my thanks and love go to my husband and partner Tim, whose understanding patience, help, encouragement and love during the course of this work and the past year has meant everything to me. Baby, I couldn't have done it without you, you were my inspiration.
iii VITA
October 19, 1958 ...... Havana, Cuba
June, 1980 ...... Wright State University, Dayton, Ohio
Sept., 1980 - Aug., 1983 ...... Laboratory Research Assistant, Wright State University
Sept., 1983 - Aug., 1985 ...... Graduate Research Assistant, Wright State University
Sept., 1986 - present ...... Graduate Research Associate, The Ohio State University, Columbus, Ohio
August 1985 ...... M.S. Polymer Chemistry, Wright State University
PUBLICATIONS
Carraher, C.E., Giron, D.J., Lopez, I., Cerutis, D.R. and Scott, W.: Platinum Polymers as Antitumoral Agents. Organic Coatings and Plastics Chemistry, 44: 120-126, 1981.
Carraher, C.E., Scott, W., Lopez, I., Giron, D.J., Cerutis, D.R. and Manek, T.: Polymeric Derivatives Based cis diamino-dichloroplatinum (II) as Antineoplastic Agents. In: Biological Activities of Polymers, ACS, Chapter 17, 221-223, eds.: C.E. Gebelein and C.E. Carraher, Plenum, N.Y., 1982.
Giron, D.J., Espy, M.J., Carraher, C.E., Lopez, I. and Turner, C.: Cell Differentiation of Platinum (II) Polyamines as Antitumor Drugs. Polymeric Materials Science and Engineering, 51: 312-315, 1984.
iv PUBLICATIONS (continued)
Carraher, C.E., Lopez, I., Giron, D.J.: Synthesis, Structural and Biological Characterization of the Polymeric Platinol Derivative of Methotrexate for the Treatment of Juvenile Diabetes. Polymeric Materials Science and Engineering, 53: 644-648, 1985.
Giron, D.J., Espy, M.J., Carraher, C.E. and Lopez, I.: Screening of Platinum (II) Polyamines as Antitumor Drugs Employing Cell Differentiation of Normal and Transformed 3T3 Cells. In: Polymeric Materials in Medication, Chapter 14, 165-172, eds.: C.E. Gebelein and C.E. Carraher, Plenum Press, N.Y., 1986.
Carraher, C.E. and Lopez, I.: Thermal Degradation and Elemental Analysis Intercorrelation for Platinum (II) Polyamines. Polymeric Materials Science and Engineering, 54: 618-622, 1986.
Carraher, C.E., Linville, R.J., Lopez, I. and Giron, D.J.: In: Advances in Biomedical Polymers: Antibacterial and Antitumoral Activity of Selected Polyphosphonoanhydrides and Polyphosphoroanhydrides. Polymer Science and Technology, ed.: C.G. Gebelein, 35, 325-333, Plenum Press, N.Y., 1987.
Carraher, C.E., Tissinger, L.G., Williams, M. and Lopez, I.: Synthesis and Characterization of Metal-Containing Enzyme Mimics. Polymeric Materials Science and Engineering, 58: 240-243, 1988.
Henningsen, G.M., Yu, K.O., Salomon, R.A., Ferry, M. J. , Lopez, I., Roberts, J. and Serve, M.P.: The Metabolism of t- Butylcyclohexane in Fischer-344 Male Rats with Hyaline Droplet Nephropathy. Toxicol. Lett., 39: 313-318, 1987.
Henningsen, G.M., Salomon, R.A., Yu, K.O., Lopez, I., Roberts, J. and Serve, M.P.: Metabolism of Nephrotoxic Isopropylcyclohexane in Male Fischer 344 Rats. J. Toxicol, and Environ. Health, 24: 19-25, 1988.
McKay, D.B., Lopez, I., Sanchez, P.A., English, J.E. and Wallace, L.J.: Characterization of Muscarinic Receptors of Bovine Adrenal Chromaffin Cells: Binding, Secretion, and Anti-Microtubule Drug Effects. Gen. Pharmac., 22(6): 1185- 1189, 1991
v FIELD OF STUDY
Major Field: Pharmacy Studies in: Neuropharmacology
vi TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
VITA ...... iv
TABLE OF ABBREVIATIONS ...... x
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xiii
CHAPTER I STATEMENT OF THE PROBLEM ...... 1
SPECIFIC A I M S ...... 3
CHAPTER II INTRODUCTION ...... 7
Adrenal Chromaffin Cells ...... 7 Physiology ...... 7 Stimulus-Secretion Coupling ...... 8
Microtubules ...... 13 Structure and Dynamics ...... 13 Posttranslational Modifications ...... 16 Microtubule-Associated Proteins ...... 18 Function of Microtubules ...... 19 Microtubule Drugs ...... 20 Microtubules in Adrenal Chromaffin C e l l s ...... 21
Neuronal nAChRs ...... 23 Structure...... 23 Pharmacology ...... 27 Neuronal nAChRs on Adrenal Chromaffin C e l l s ...... 29
Receptor-Cytoskeleton Associations ...... 31
vii CHAPTER III Materials and Methods 37
M a t e r i a l s ...... 37 Chemicals and Antibodies ...... 37 Cell Culture Supplies ...... 38
M e t h o d s ...... 39 Adrenal Chromaffin Cell Isolation ...... 39 Catecholamine Secretion Studies ...... 47 Loading Cells with [3H]Norepinephrine ...... 47 Catecholamine Secretion Experiments .... 48 Tubulin Immunocytochemistry ...... 50 General Procedure ...... 50 Acetylated Tubulin Studies ...... 53 Rat Brain Membrane Preparation ...... 53 [3H]Nicotine Binding to Rat Brain Membrane Fragments ...... 54 [3H] Nicotine Binding to Intact Adrenal Chromaffin Cells ...... 55 Growth and Maintenance of mAb 3 5 Secreting Hybridoma Cell L i n e ...... 56 Purification of mAb 3 5 ...... 57 Iodination of mAb 3 5 ...... 59 [125I]mAb 35 Binding to Intact Adrenal Chromaffin Cells ...... 59 [125I]mAb 35 Binding to Triton-Extracted Adrenal Chromaffin Cells ...... 60 Calculations and S t a t i s t i c s ...... 61
CHAPTER IV R e s u l t s ...... 62
Effects of Microtubule Drugs on Nicotine- Stimulated [3H]Norepinephrine Release from Adrenal Chromaffin Cells ...... 62 Effects of Microtubule Drugs on Catecholamine Secretion Mediated by Various Noncholinergic Secretagogues ...... 62 Effects of Microtubule Drug Treatment Time on Nicotine-Stimulated Catecholamine Secretion ...... 65 Reversal of the Effects of the Microtubule Drugs on Nicotine-Stimulated Catecholamine R e l e a s e ...... 69 Effect of Chronic Treatment with Microtubule Drugs on Adrenal Catecholamine R e l e a s e ...... 71 Effects of Microtubule Drugs on the Microtubular Network of Adrenal Chromaffin C e l l s ...... 73 Time-Course for the Appearance of Vinblastine- Induced Paracrystals ...... 80
viii Effect of Microtubule Drugs on the Acetylated Tubulin Population of Adrenal Chromaffin C e l l s ...... 88 Effect of Microtubule Drugs on [3H]Nicotine Binding to Rat Brain Membrane Fragments .. . 95 Effect of Microtubule Drugs on [3H]Nicotine Binding to nAChRs in Intact Adrenal Chromaffin Cells ...... 98 Noncompetitive Inhibition of Nicotine- Stimulated Catecholamine Release by by Microtubule Drugs ...... 100 Effect of Triton-Extraction on [125I]mAb 35 Binding to Adrenal Chromaffin Cells...... 103 Influence of Microtubule Drugs on the Association of adrenal nAChRs to Triton- Extracted Adrenal Chromaffin Cells...... 108 Effects of Microtubule Drugs on [125I]mAb 35 Binding to Intact Adrenal Chromaffin Cells . Ill
CHAPTER V DISCUSSION ...... 113
CHAPTER VI SUMMARY AND CONCLUSIONS ...... 126
REFERENCES ...... 128
ix TABLE OF ABBREVIATIONS
ABBREVIATION NAME
ACh ...... Acetylcholine
ara-C ...... Cytosine Arabino-furanoside
a-Bgt ...... alpha-Bungarotoxin
BSA ...... Bovine Serum Albumin
Ca+ 2 ...... Calcium
c m ...... Centimeter
c p m ...... Counts Per Minute
C i ...... Curie
DNA ...... Deoxyribonucleic Acid
DMSO ...... Dimethylsulfoxide
dpm ...... Disintegrations Per Minute
DMEM ...... Dulblecco's Modified Eagle's Media
D-PBS ...... Dulbecco's Phosphate Buffered Saline
EDTA ...... Disodium ethylenediamine- tetraacetate
FDU ...... 5-Flurodeoxyuridine
FITC ...... Fluorescein Isothiocyanate
HEPES ...... N-[2-hydroxyethyl] piperazine- N'-[2-ethanesulfonic acid] h r ...... hour
x TABLE OF ABBREVIATIONS (continued)
ABBREVIATION NAME
125I ...... Iodine 125
L ...... Liter
jxCi...... MicroCurie
/ i g ...... Microgram
H i ...... Microliter
/ x M ...... Micromolar
m g ...... Milligram
m l ...... Milliliter
m M ...... Millimolar
m m ...... Millimole
m i n ...... Minute
M ...... Molar (moles/liter)
mAb ...... monoclonal antibody
nAChR ...... Nicotinic Acetylcholine Receptor
n M ...... Nanomolar
PFHM ...... Protein Free Hybridoma Media
PMSF ...... Phenylmethylsulfonyl Fluoride
PSS ...... Physiological Salt Solution rpm ...... Revolution Per Minute s e c ...... Second
TCA ...... Trichloroacetic Acid v / v ...... Volume by Volume w / v ...... Weight by Volume
XI LIST OP TABLES
TABLE PAGE
1. Comparison of IC50 values of various microtubule drugs for inhibition of nicotine-stimulated catecholamine release and for inhibition of in vitro tubulin polymerization ...... 63
2. Selective inhibtion of nAChR-stimulated adrenal catecholamine release by microtubule drugs ...... 66
3. Effects of microtubule drugs on basal (nonstimulated) catecholamine release ...... 67
4. Effects of 24 hr treatment with low concentrations of microtubule drugs on basal (nonstimulated) catecholamine release ...... 74
5. Effects of microtubule drugs on the concentration-response effects of nicotine on adrenal catecholamine release ...... 101
6. [125I]mAb 3 5 binding to Triton-extracted cultured adrenal chromaffin cells ...... 106
7. Effects of microtubule drugs on [125I]mAb 35 bound to Triton-extracted adrenal chromaffin cells ...... 109
8. Effects of microtubule drugs on [125I]mAb 35 binding to cultured adrenal chromaffin cells .... 112 LIST OF FIGURES
FIGURE PAGE
1. Schematic representation of neuronal nAChRs association with cytoskeletal components ...... 4
2. Structure of microtubule drugs ...... 5
3. Perfusion apparatus ...... 41
4. The concentration-response effects of several microtubule drugs on nAChR-stimulated adrenal catecholamine release ...... 64
5. Effects of various microtubule drugs on [3H]norepinephrine release stimulated by 10 /iM nicotine or 500 nM histamine in cultured bovine adrenal chromaffin cells ...... 68
6. Time-dependent effects of microtubule drugs on nAChRs-stimulated [3H]norepinephrine release . . 70
7. Reversal of the effects of microtubule drugs on nAChR-stimulated [3H]norepinephrine release from cultured bovine adrenal chromaffin cells. . . . 72
8. Effects of chronic treatment with microtubule drugs on nicotine- and K+-stimulated [3H]norepinephrine release...... 75
9. Effects of a 60 min treatment with various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-tubulin antibodies ...... 76
xiii FIGURE PAGE
10. Effects of a 180 min treatment with various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-tubulin antibodies ...... 81
11. Time-course for the appearance of vinblastine- induced paracrystals in cultured adrenal chromaffin cells by anti-tubulin antibodies . . 85
12. Effects of various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-acetylated tubulin antibodies (6-11B-1) 90
13. Drug displacement curves of specific [3H]nicotine binding by unlabelled nicotine and carbachol ...... 96
14. Effects of various microtubule drugs on [3H]nicotine binding in rat brain membrane f r a g m e n t s ...... 97
15. The effects of various microtubule drugs on [3H]nicotine binding to intact primary cultures of adrenal chromaffin cells ...... 99
16. The concentration-response effects of nicotine on adrenal catecholamine release in the absence and presence of various microtubule drugs ...... 102
17. Effect of 24 hr exposure with mAb 35 on secretagogue-stimulated adrenal [3H]norepinephrine release ...... 105
18. [125H]mAb 35 binding to Triton-extracted cultured adrenal chromaffin cells ...... 107
19. Effect of microtubule drugs on [125I]mAb 35 binding to Triton-extracted adrenal
xiv The overly cautious scientist neither errs nor discovers W.I.B. Beveridge CHAPTER I
STATEMENT OF THE PROBLEM
Microtubules are major components of the neuronal
cytoskeleton (Peters et al., 1976; Baas and Black, 1990).
They have been implicated in a variety of cellular functions
including secretion (Burgoyne, 1991; Schroer and Sheetz,
1991). This is based mainly on the ability of colchicine and vinblastine, drugs which interfere with the function of microtubules, to inhibit secretory processes (Poisner, 1973;
Cooke and Poisner, 1979; Poisner and Bernstein, 1971; Lacey et a l., 1968) .
In adrenal chromaffin cells, microtubule drugs such as taxol and vinblastine, inhibit nicotine-mediated catecholamine release without affecting release stimulated by other secretagogue (Trifaro et a l ., 1972; McKay and
Schneider, 1984). McKay and coworkers (1984) have also demonstrated that vinblastine interferes with histrionicotoxin binding to sites associated with the nicotinic acetylcholine receptor (nAChR) ion channel complex from Torpedo electric organ. These results suggest that taxol and vinblastine are acting at the level of the nAChR to interfere with receptor function and binding. These
1 2
studies together suggest that the action of the microtubule
drugs is not on the terminal steps of exocytosis, but
instead alter nAChR function by either a direct effect of
the drugs on the receptor or an effect on microtubule links
to the nAChRs of adrenal chromaffin cells, suggesting
adrenal nAChRs are associated with the cytoskeleton.
Another possibility is that microtubule drugs may possess
anti-cholinergic activity (Trifaro et. al., 1972; McKay and
Schneider, 1984)
Numerous membrane receptors have been demonstrated to be associated with the cytoskeletal network, including receptors for nerve growth factor (Vale and Shooter, 1982) ,
Platelet-Derived Growth Factor (Zippel et al., 1989), transferrin (Hunt et al., 1989) and fibronectin (Horwitz et a l ., 1986). Association of these receptors with the cytoskeleton has been demonstrated to play an important role in receptor function. Receptor-cytoskeletal associations have also been demonstrated for muscle nAChRs (Prives, et al., 1982; Stya and Axelrod, 1983; Connolly and Oldfin,
1985). In muscle, microtubules may participate in anchoring receptors (Connolly and Oldfin, 1985).
Evidence in the literature suggests that adrenal nAChRs are associated with microtubular protein. This is based primarily on the ability of vinblastine and taxol to inhibit nAChR-stimulated secretion (McKay and Schneider, 1984; McKay et a l ., 1985). Therefore, if indeed microtubules play a role in adrenal nAChR function, then other microtubule drugs should have the same or similar effects on the function of adrenal nAChRs and should affect adrenal microtubules under the same or similar conditions that affect receptor function. The hypothesis under investigation is that adrenal nAChRs are associated with microtubules and that this association may be important for receptor function. A schematic representation of this hypothesis is found in Fig.
1. The major objectives of this project were to determine
1) the site and mechanism of action of various microtubule drugs (Fig. 2) on adrenal nAChR-stimulated catecholamine release and 2) to determine if adrenal nAChRs are associated with microtubules.
Specific Aims
1. To determine the effects various microtubule drugs,
have on nAChRs-mediated adrenal catecholamine release.
2. To determine if microtubule drugs block secretion
mediated by non-nAChR mechanisms.
3. To determine if microtubule drugs alter chromaffin cell
microtubular network under similar conditions shown to
affect nAChR function (secretion).
4. To determine if adrenal chromaffin cells contain
acetylated microtubules.
5. To determine if microtubule drugs alter the acetylated
microtubular network under conditions shown to affect 4
Figure 1. Schematic representation of adrenal nAChRs associated with cytoskeletal components. Microtubules (MTS), Microtubule-Associated Proteins (MAPS). Adapted from Hucho, 1986). 0— C - CM, I 0
Tfftol
OH ir
,T
CH. OH
R 3H,0 * 0R 3CH CH jO OCHj R l C - R 2
VlnblMlln*: Rl - CHj: R2 - OCHj: R3 - COCHj Vlncrtttfna: Rl - CHO: R2 - OCHj; R3 - COCHj Vlnd«*inr Rl - CHj; R2 - NHj; R3 - H Podoohylloloxin: Rl =* R2 » H R3 = OH: R4 = CHj
CH,0 KHCH
CH-0
OCTl
•Demecolcine
CH
CH-S NH-C-CH.-CH
Tiibulaiole (It If! Rlfi).
Figure 2 Chemical structure of various microtubule drugs, 6
nAChRs function.
6. To determine the time-dependent effects of the
microtubule drugs on nicotine-stimulated catecholamine
secretion.
7. To determine if the effects of the drugs on nicotine
mediated release are reversible.
8. To determine if the anticholinergic effect of the
microtubule drugs is due to an interaction with the
nicotine binding site.
9. To determine if adrenal nAChRs are associated with the
Triton-insoluble cytoskeleton.
10. To determine if microtubule drugs alter the association
of nAChRs to the Triton-insoluble cytoskeleton. CHAPTER II
INTRODUCTION
ADRENAL CHROMAFFIN CELLS
Physiology
The adrenal medulla is an endocrine gland. Secretory
products released by the adrenal medulla help in the
regulation of involuntary functions such as heart rate,
intestinal motility and pupillary dilation. At birth it is
comprised of primitive sympathetic cells which differentiate
into chromaffin cells (pheochromocytes). Chromaffin cells
synthesize, store and secrete a complex mixture containing
epinephrine, norepinephrine and a variety of other proteins
and peptides such as enkephalins, chromogranins and
neuropeptides (Bank et al., 1965; Douglas et a l ., 1965;
Aunis et al., 1980; Viveros et al., 1983,; Livett, 1984).
Adrenal chromaffin cells are homologous to sympathetic
neurons. Both types of cells are derived from the neural crest, synthesize and release catecholamines, receive synaptic input from preganglionic cholinergic neurons and are stimulated by the activation of nicotinic acetylcholine receptors (nAChRs). Chromaffin cells also share common subcellular features with many neuronal cells. One such
7 8
feature is the storage of secretory products in membrane
bound granules. In culture, these secretory cells have been
referred to as paraneurons, a term that includes cells generally and traditionally not considered neurons and yet regarded as relatives of neurons on the basis of their structure and function (Trifaro, 1983, 1989). When chromaffin cells are grown in culture, they extend axon-like processes, giving the cells a neuronal appearance (Unsiker, et a l., 1980; Trifaro et al., 1980; Livett, 1984).
In almost all species, chromaffin cells possess both nicotinic and muscarinic receptors which can stimulate catecholamine secretion (Douglas and Poisner, 1965).
However, in some species (e.g., rat chromaffin cells; Brandt et al., 1976), secretion is mediated by the activation of muscarinic receptors while in others (e.g., bovine; Livett,
1983) only stimulation of nicotinic receptors will elicit secretion. Other receptors present on chromaffin cells which elicit release of catecholamines include angiotensin
II (Bunn and Marley, 1989), VIP (Wilson, 1988), bradykinin
(O'Sullivan and Burgoyne, 1989; Plevin and Boarder, 1988), histamine (O'Sullivan and Burgoyne, 1989; Chaliss, et a l .,
1991) and prostaglandin E2 (Ito, et al., 1991).
Catecholamine Secretion Studies
Activation of chromaffin cell nAChRs by the neurotransmitter, acetylcholine (ACh), leads to an inward 9
flux of Na+ and to a lesser extent Ca+2 through the nAChR-
associated ion channel (Douglas, et al., 1967). This
results in a slight depolarization of the cell membrane,
sufficient to activate the voltage-sensitive fast Na+
channels (Douglas, et al., 1967; Kidokoro, et al., 1982).
Opening of both the receptor ion channel and the voltage
sensitive fast Na+ channels allows influx of Ca+2
(Kilpatrick et al., 1981; Corcoran et al., 1983) and a
concomitant increase in the concentration of intracellular
Ca+2.- This increase in intracellular Ca+2 is the primary
stimulus for exocytosis of chromaffin granules containing
catecholamines (Kilpatrick et al., 1982; Burgoyne et al.,
1982) . The molecular mechanism involved in changing the
Ca+2 signal into the mechanics of granule fusion with the
cell membrane and release of the granule contents is not
known. Current advances in stimulus-secretion coupling
suggest the involvement of Ca+2 binding proteins (eg. ,
calmodulin, calpactin, synexin), Ca+2 dependent kinases
(protein kinase C), cytoskeletal elements and their
associated proteins, guanosine nucleotide binding proteins
as well as plasma membrane and granule-associated proteins
(Marley, 1988; Burgoyne 1991).
Catecholamine secretion can also be elicited with high extracellular K+ (Baker and Rink, 1975) or by using a Ca+2 selective ionophore [e.g., calcimycin (A23187), Burgoyne,
1984]. Depolarization by excess K+ bypasses the nicotinic 10
receptor and directly activates the voltage-sensitive Na+
channel. K+-induced secretion is also sensitive to
extracellular Ca+2 and can be blocked by Ca+2 channel
antagonists such as verapamil (Kidokoro, et al., 1982).
Catecholamine release in chromaffin cells will progressively decline if the cells are continuously exposed to nicotinic agonists (Boska and Levitt, 1984; Boska and
Levitt, 1984b). Previous studies by Sasakawa and coworkers
(1985) have shown that pretreatment of cells with nicotinic agonists had no effect on K+-induced Ca+2 influx. Their results suggest that the loss of sensitivity to nicotinic agonists was due to receptor desensitization instead of inactivation of Ca+2 channels. Furthermore, low concentrations of substance P have been shown to protect the nicotinic response from desensitization without affecting inactivation of K+-induced catecholamine secretion (Boska and Levitt, 1984). These results indicate that desensitization can also occur at the level of the nicotinic receptor.
Mechanisms involved in secretion are very similar between secretory cells (i.e., exocrine, endocrine and neuronal cells). Many cells possess a regulated secretory pathway (Trifaro, 1977). This pathway involves the concentration and packaging of material to be exported into storage granules and transport of the vesicles to the site of release via microtubule-medj.ated transport. The 11
molecular mechanisms involved in the late stages of
secretion, such as the movement of vesicles to the inner
surface of the plasma membrane, docking of the vesicles on
the plasma membrane and exocytotic membrane fusion and
fission remain unknown.
In endocrine cells, the vesicles are prevented from
reaching the plasma membrane prior to stimulation (Burgoyne
and Cheek, 1987; Orci, 1972). Studies by Kondo et a l .
(1982) have demonstrated that the secretory granules of
adrenal chromaffin cells are embedded in a three-dimensional
cytoskeletal network. This suggests that an association
between secretory granules and the cytoskeleton could be
important in the regulation of exocytosis, either by holding
vesicles at the plasma membrane ready to be released or by
preventing access of the secretory granules to the plasma
membrane until the cell is stimulated, as has been suggested
in adrenal chromaffin cells (Trifaro et al., 1983; Burgoyne
et a l ., 1986).
Much of the evidence acquired for the involvement of
cytoskeletal elements in secretion has been gathered from adrenal chromaffin cells. Additionally, adrenal chromaffin cells have been used to study the interactions of cytoskeletal proteins with secretory vesicles (Burgoyne,
1987; Trifaro, 1989). Among the cytoskeletal proteins involved in transporting granules from the Golgi network to their site of release and maintaining the vesicles near 12
exocytosis sites are the cytoskeletal elements, microtubule
proteins (discussed below) and actin. The cytoskeletal
elements that interact with vesicles and impair their
movement are believed to be different from microtubules and
are thought to be mainly actin, caldesmon and fodrin
(Koideli, 1986).
Actin binds to granule membranes through actin binding
proteins such as a-actinin, fodrin and caldesmon. These
proteins are extrinsic components of the secretory granule
membrane. It has been demonstrated that binding of actin to
secretory granule membranes, as has been observed in
chromaffin granules, is increased at nM concentrations of
Ca+2, resulting in the formation of a crosslinked gel
(Burgoyne et al ., 1982; Trifaro et al ., 1983). This
gelation increases the viscosity of the cytoplasm, and thus
serves to dock the vesicles at the plasma membrane in
preparation for exocytosis. Formation of this actin network
gel can be inhibited by micromolar concentrations of Ca+2
(Stendahl et al ., 1980), thus providing a mechanism to
control granule exocytosis. It has been demonstrated in
vitro that upon cell stimulation, intracellular Ca+2
concentration increases which leads to the release of
secretory granules from the actin cytoskeleton, thus enabling the granules to interact with the plasma membrane or plasma membrane cytoskeleton and to undergo exocytosis
(Burgoyne et al., 1982). Thus the assembly/disassembly of 13
actin may play an important role in the mechanism of
exocytosis.
MICROTUBULES
Structure and Dynamics
Microtubules are widely distributed in eukaryotic
cells, particularly nerve cells (Borisy and Taylor, 1967;
Feit et al., 1971). The major protein constituent of
microtubules is tubulin, a heterodimer composed of a- and R-
subunits (Bryan and Wilson, 1971; Luduena et a l ., 1964,
1981). In addition to tubulin, microtubules contain a
heterogeneous class of proteins referred to as microtubule
associated proteins (MAPs), which promote tubulin assembly
and affect the stability of polymerized microtubules (Black,
1986). Microtubules play a role in a number of important
and diverse cellular processes such as cell division,
development and maintenance of cell morphology, cell motility and intracellular transport and exocytosis (Fulton,
1981; Manfredi and Horwitz, 1984; Avila, 1990).
Microtubules are slender, hollow tubules about 25 nm
(20-27 nm) in diameter. They are unbranching and appear to have a certain amount of rigidity, but at the same time have enough elasticity to bend. In cross section, they appear to be made up of 13 subunits which are arranged in parallel, longitudinal rows (Dustin, 1984). Microtubules are composed of two protein monomers of identical molecular weight 14
(54,000 daltons), a- and 6 -tubulin, which are present
throughout the cytoplasm (Dustin, 1984). The two tubulins
form dimers which in turn polymerize to form the
longitudinal parallel strand protofilaments. This
polymerization requires Mg+2, GTP and MAPs (Manfredi and
Horwitz, 1984; Avila, 1990). Within each microtubule, a-
and 6 -tubulin are arranged in a helical pattern. It has
been suggested that MAPs may be involved with regulation of
polymerization (Black, 1986).
Many of the functions performed by microtubules depend
on their ability to polymerize and depolymerize. These
processes can be blocked by agents that prevent microtubule
assembly or disassembly (discussed below). In order to
better understand how microtubule drugs may affect these
processes, it is important to understand the mechanism
involved in microtubule dynamics. Formation of microtubules
involves two stages, initiation and elongation (Weisenberg and Deery, 1976; Erickson and Voter, 1986). Initiation appears to involve formation of a ring-like structure to which tubulin subunits can be added. In vivo, initiation is still not well understood; however, microtubule formation appears to occur around microtubule organizing centers
(MTOC), such as centrioles (Brinkley et al., 1981; Erickson and Voter, 1986) .
Microtubules elongation occurs by incorporation of tubulin monomers to one end of the microtubule. The two ends of the filament are termed the plus and minus ends,
based on relative rates of tubulin polymerization. The plus
end is the rapidly polymerizing end, whereas, the minus end
polymerizes at a much slower rate (Mitchison and Kirschner,
1984) . Addition of tubulin dimers to the growing end of the
microtubule is followed by a delayed GTP hydrolysis.
However, tubulin polymerization is not dependent on GTP
hydrolysis. If tubulin molecules are being added to the
polymer end faster than GTP hydrolysis, a GTP cap will form
at the growing end which stabilizes the growing microtubule
(Mitcheson and Kirschner, 1984). Since tubulin-GTP
complexes bind to each other with greater affinity, the molecules will continue to grow, contributing to the population in the growing phase. On the other hand, if polymerization slows down, GTP hydrolysis to GDP occurs, resulting in a rapid and complete depolymerization of the microtubule (Mitcheson and Kirschner, 1984).
As cells grow and differentiate, two populations of microtubules appear: a dynamic (labile) population and a stable population (Schulze and Kirschner, 1987). Dynamic microtubules can be disrupted by depolymerization with drugs such as demecolcine, nocodazole or colchicine (Schulze and
Kirschner, 1987). stable microtubules are more resistant to disruption by some microtubule drugs (e.g., colchicine).
Dynamic microtubules are characteristic of cells undergoing an internal reorganization such as constantly dividing cells 16
or migrating cells. Stable microtubules are more prominent
in highly differentiated cells, such as neurons, which have
lost their ability to divide (Brady et al ., 1984). In these
cells, the microtubules become permanent features, i.e.,
they are essentially nondynamic, neither growing nor
shrinking appreciably (Sahenk and Brady, 1987). The exact
mechanism by which microtubules are stabilized is not known,
although it has been suggested that posttranslational
modifications of the microtubule protein or interaction of
microtubules with certain microtubule-associated proteins
(MAPs) may play a role (Hamel et al ., 1983; Matus, 1988).
Posttranslational Modifications of Microtubules.
Although much work has been done on posttranslational
modifications of tubulin, little is still known regarding
the in vivo role of these modifications. It has been
suggested that these modifications may act as signals for the binding of specific MAPs that stabilize microtubules and modulate the properties of the microtubule network in response to changes in the internal or external milieu
(Black and Keyser, 1987; Piperno et a l., 1987).
Microtubules can undergo at least three types of posttranslational modifications: tyrosinylation, acetylation and phosphorylation. The first posttranslational modification described for tubulin was tyrosination/detyrosination (Berra et al., 1988; Gunderson 17
et a l ., 1984, 1987). In this type of modification, a
tyrosine residue is inserted at the carboxy terminus of its
a-chain by a tyrosine ligase or is released via a
carboxypeptidase (Berra et al., 1988). Tubulin can also be
modified by phosphorylation both in vivo and in vitro (Gard
and Kirschner, 1985; Diaz-Nido et a l ., 1990).
Acetylation of a-tubulin is another form of posttranslational modification. This form of tubulin modification was first observed in Chlamydomonas during
flagellar regeneration (L'Hernault and Rosenbaum, 1985).
The availability of a monoclonal antibody, 6-11B-1, which specifically recognizes the acetylated form of tubulin, has facilitated the study of this posttranslational modification
(Piperno and Fuller, 1985). Acetylated microtubules are more resistant to depolymerization by microtubule drugs such as colchicine and nocodazole (Piperno et a l ., 1987; Falconer et al., 1989). This type of microtubule is prominent in neurons (Black and Keyser, 1987). The enzyme, tubulin acetyltransferase, acetylates the e-amino group of a lysine residue in a-tubulin. Acetylation probably represents a modification of preformed microtubules, since it is inhibited by microtubule depolymerizing drugs in cultured neuronal cells. In fact, the tubulin polymer is a better substrate than the dimer for a-tubulin acetyltransferase.
Acetylated microtubules comprise 10 - 40 % of the total cytoplasmic microtubule network, although exceptions exist in which few or none are found (Maruta et al., 1986). In
most instances, stable microtubules containing acetylated
tubulin colocalize with detyrosinated tubulin, but the
distribution and extent of each modification is not always
identical.
Microtubule-Associated Proteins (MAPs)
MAPs are a group of heterogenous phosphoproteins
originally identified as brain proteins that co-purify with
tubulin through several cycles of temperature-dependent
assembly and disassembly cycles (Olmsted, 1987; Valle et
a l ., 1987). These proteins stabilize assembled microtubules
and are involved in modulating tubulin polymerization into
microtubules (Olmsted, 1987; Lewis et al., 1989; Cross et
al., 1991). These functions are mediated by specific
interactions of MAPs with tubulin. MAPs are divided into
two major classes based on their molecular weight: a group
of high molecular weight proteins, known as MAPI and MAP2,
and a group of 50 to 7 0 kD proteins, referred to as tau
factors (Olmsted, 1987). Both classes of MAPs have two
domains. One domain binds microtubules. The other domain
is thought to be involved in linking microtubules to other cytoskeletal components (actin-filaments, neurofilaments) and cellular organelles (Olmsted, 1987). Other MAPs have also been identified and it has been suggested that some of these MAPs play a structural role in stabilizing 19
microtubules, in linking microtubules to other cellular
organelles and in transporting organelles along
microtubules.
Function of Microtubules
In various secretory systems, microtubule-directed
transport plays an important role in the intracellular
movement of organelles. For example, in mast cells,
movement of histamine granules is directed by microtubules
(Gillespie et al., 1968). Similarly, the insulin-producing
fi cells of the islets of Langerhans have numerous
microtubules which direct alignment of granules into rows
near the cell surface (Lacey et al., 1968). Exocytosis of
these granules is not affected by microtubule disrupting
drugs. However, it has been demonstrated that colchicine
inhibits the secondary or slower phase of insulin release, which suggests movement of the secretory granules to release sites is dependent on microtubules. This mechanism of secretion applies to many secretory cells.
In neurons, where the sites specialized for exocytosis are usually very distant from the cell body, microtubule transport is very important (Schroer and Sheetz, 1991) .
Microtubules are also necessary for the extension and maintenance of neuronal processes and serve as tracks for axoplasmic flow (Vale, 1987). Electron micrographs show associations between organelles presumably being transported 20
by microtubules (Allen et al., 1987; Allen, 1987). It has
been demonstrated that a single microtubule can support
organelle movement in both directions and can carry small
vesicles and mitochondria throughout the cytoplasm (Hayden
et al, 1984; Schnapp et al., 1985; Vale, 1987). The squid
giant axon has been extensively used to study microtubule-
mediated transport. Examination of extruded axoplasm from
this axon by video-enhanced light microscopy has shown
organelles moving along filamentous tracks (Vale, 1987).
The use of immunofluorescent techniques and electron
microscopy have demonstrated that these tracks are single
microtubules (Vale, 1987; Brady et al., 1982). In neurons,
microtubule transport is important for targeting secretory
vesicles to their sites of release.
Microtubule Drugs
To evaluate the role of microtubules in the transport
of secretory vesicles to the cell surface and in exocytosis,
most studies have employed the use of microtubule disrupting
drugs such as colchicine, the vinca alkaloids (vinblastine
and vincristine), demecolcine (colcemide) or podophyllotoxin
or the microtubule stabilizing drug, taxol, to interfere with secretion. Colchicine, podophyllotoxin and demecolcine alter microtubules by binding to free tubulin which prevents
further addition of tubulin to microtubules, leading to microtubule depolymerization. At high concentrations, the 21
vinca alkaloids disrupt microtubules by inducing the
formation of paracrystalline aggregates of tubulin. Taxol
has the opposite effect; it binds to microtubules and
enhances assembly of free tubulin into microtubules,
preventing depolymerization.
In a variety of cells including fibroblasts,
hepatocytes, mammary gland cells and exocrine and endocrine
cells, disruption of microtubules with microtubule drugs
will alter secretion. It has been observed that release of
secretory granules made prior to drug treatment and that lie
at the cell surface is usually not affected. However, it
has been demonstrated that secretion of newly synthesized materials is inhibited, suggesting that microtubules are
required for transport of these proteins to the surface.
Microtubules in Adrenal Chromaffin Cells
In chromaffin cells, cytoskeletal elements such as microtubules, actin) and their regulatory proteins have been isolated and characterized (Trifaro, 1977; Bader et a l .,
1984). Indirect immunofluorescence techniques using antibodies raised against tubulin have localized the distribution of the microtubule network; microtubules are found in the cell body and along the neurites. Early work by Poisner and Bernstein (1971), using the microtubule disrupting drugs colchicine, vinblastine and vincristine, suggested that microtubules were involved in the secretory 22
mechanisms of chromaffin cells. Bader and coworkers (1984)
demonstrated that treatment of chromaffin cells with
colchicine induced depolymerization of microtubules and a
return of secretory granules located at the tip of neurites
to the cell body, which was followed by a complete
retraction of the neurites. These results supported a role
for microtubules in the transport of secretory granules and
in neurite outgrowth and maintenance. High affinity binding
sites for tubulin have been demonstrated on chromaffin
granules, providing further evidence for an interaction
between secretory granules and microtubules (Trifaro, 1978).
Studies conducted by Trifaro and colleagues (1972)
indicated that, although microtubules might be involved in
transport of chromaffin granules to the cell periphery, they
were not involved in the final steps of secretion. This was
based on the fact that exocytosis of catecholamines induced
by depolarizing concentrations of K+ were not blocked by
either colchicine or vinblastine. These studies suggest
that the effect of colchicine and vinblastine on secretion were due to an anticholinergic action of the drugs (Trifaro et al., 1972). Other studies employing the microtubule drugs taxol, colchicine and vinblastine have also demonstrated a selective inhibitory effect on ACh stimulated catecholamine release from cultured adrenal chromaffin cells
(McKay and Schneider, 1984). In these studies it was also shown that the microtubule drugs do not block catecholamine 23
secretion induced by other agents such as depolarizing
concentrations of K+, veratridine or barium (McKay and
Schneider, 1984). Therefore, in adrenal chromaffin cells,
microtubules may play a role in transport of secretory
granules to their site of release, but they are not involved
in the terminal steps of a common secretory pathway.
NEURONAL nAChRs
nAChRs are found in neuromuscular junctions and on
neuronal tissues (sympathetic ganglia, brain and adrenal
medulla). Although muscle-type nAChRs are fairly well
characterized, less is known about adrenal nAChRs and other
neuronal-type nAChRs. Pharmacological similiarities between
neuronal and muscle nAChRs have been reported (Lipscomb and
Rang, 1988). Other studies though, have indicated that
these two receptor types differ in their sensitivities to
various nAChR antagonists and neurotoxins (Galzi et al.,
1991). It is also becoming clear that neuronal nAChRs
differ from muscle nAChRs in both structure and regulation
(Galzi et al., 1991).
Structure
Nicotinic cholinergic transmission occurs in the
central nervous system, autonomic ganglia and adrenal medulla (Clarke et al., 1984; Clarke et al., 1985; Smith et a l . nervous system were based on a-Bgt binding to brain 24
membranes. However, this toxin was not a useful tool for
these studies since it did not block ACh-induced ion
conductance in the CNS (Oswald and Freeman, 1981; Morley and
Kemp, 1981; Clark et al ., 1985; Marks et al., 1986). It
does not bind to brain [3H]nicotine or [3H]ACh-binding sites
(Patrick and Stallcup, 1977; Clark et a l ., 1985) or compete
for nicotinic binding sites in rat brain membranes (Romano
and Goldstein, 1980). Studies by various groups suggest
that a-Bgt in brain binds to a pharmacologically distinct
subtype of neuronal nicotinic receptor (Couturier et a l .,
1990; Schoepfer et a l., 1990).
Pharmacological characterization of neuronal nAChRs has
been facilitated by a new toxin, neuronal bungarotoxin or
bungarotoxin 3.1 (Bgt 3.1). Bgt 3.1 has been shown to block
nicotinic cholinergic transmission in chick ciliary ganglion
and sympathetic ganglia (Chiappinelli, 1983; Chiappinelli
et a l ., 1985). Affinity labeling studies using Bgt 3.1 have
identified a 59 kD putative nAChR subunit in chick ciliary
ganglia (Halvorsen and Berg, 1987); similarly, a putative
neuronal nAChR that binds mAb 35 and Bgt 3.1 has been
identified on bovine chromaffin cells (Higgins and Berg,
1987).
Another probe for identifying nAChRs in neuronal tissue has been of anti-nAChR monoclonal antibodies (Whiting et a l ., 1987). A monoclonal antibody (mAb 35) directed against the main immunogenic region of the Torpedo nAChR a subunit 25
was found to cross-react with a component in chick ciliary
ganglia (Jacob et al., 1984; Smith et al ., 1985). Using mAb
35, a putative chick brain nAChR was isolated and found to
be contain two proteins of 49 and 58 kD (Whiting and
Lindstrom, 1987a). The purified receptor from chick brain
was then used to raise another monoclonal antibody (mAb 270)
which cross reacted with a rat brain component (Whiting and
Lindstrom, 1987b). mAb 270 has been used to isolate a
putative neuronal nAChR from rat brain containing two
different proteins with molecular weights of 51 and 79 kD
(Whiting and Lindstrom, 1987a, 1987b). The 49 and 58 kD proteins from chick brain and the 51 and 79 kD proteins from rat brain were designated as neuronal nAChR a and B subunits, for each species (Smith et al ., 1985; Whiting and
Lindstrom, 1987a, 1987b).
These proteins purified from brain are referred to as a and 6 based on molecular weight and cross reactivity with subunit-specific antibodies raised against Torpedo nAChRs
(Whiting and Lindstrom, 1986). However, when these purified subunits from chick or rat brain were incubated with the ACh affinity analog, 4-(N-maleimido)benzyl-trimethylammonium
(MBTA), which affinity labels the a subunit of Torpedo and muscle AChRs, only the B subunit was labeled (Whiting and
Lindstrom, 1987b). From these studies it was concluded that the B subunit from brain contains the ACh-binding site and was more closely related to the a subunit of Torpedo and muscle nAChR. Therefore, the terminology was changed: the
51 and 79 kD proteins were designated as the a subunit and
the 49 and 58 kD proteins as B subunit. Whiting and
Lindstrom (1986) first designated the stoichiometry for
these receptors as a2®3 * T*ie ProPosed <*263 stoichiometry has been accepted since it is analogous to that of the nAChR
from Torpedo electric organ and muscle with two identical
ligand binding subunits in a total of five subunits (Whiting
et al., 1987a, Whiting and Lindstrom, 1987b). Studies by
Cooper and coworkers (1991) have also demonstrated that
functional neuronal nAChRs are pentameric complexes composed
of two a and three non-a (net, B or structural) subunits.
Several lines of evidence indicate that neuronal nAChRs are made up of only two subunits, an a subunit which binds agonists and a 6 (structural) subunit (Whiting and
Lindstrom, 1987b; Bertrand et al., 1990; Boulter, 1990).
Molecular techniques have also contributed to the identification of neuronal nAChRs subunits. Expression of cDNAs for the structural subunits and the ACh-binding subunits in Xenopus oocytes produces functional AChRs
(Boulter et al., 1987). Analysis of the cDNA sequences for subunits of neuronal nAChRs demonstrates the presence of four hydrophobic domains which correspond to those of muscle nAChR (Boulter et al., 1987). The amino acid sequence of the second hydrophobic domain is the most conserved
(Lindstrom et al., 1989). All subunits of neuronal nAChRs 27 have an N-glycosylation site and cysteine residues
corresponding to those found on the muscle nAChR a subunit
(al92-193), which are characteristic of binding subunits and
accounts for their MBTA reactivity (Tzartos et a l . , 1988;
Das et al., 1989; Schoepfer et al ., 1989; Fronasari et a l .,
1990). All of the subunits in neuronal nAChRs contain a
long hydrophilic domain of variable length, located at C-
terminal of M3. This region corresponds to nonhomologous
highly immunogenic sequences on the cytoplasmic surface of
muscle nAChRs (Ratnam et al., 1986; Schoepfer et a l ., 1989).
It has been proposed that this cytoplasmic loop, as well as
other cytoplasmic domains, like muscle, might interact with cytoskeletal elements to anchor the nAChRs at their functional locations (Schoepfer et al., 1989).
Pharmacology
Molecular and electrophysiological techniques have contributed in identifying several genes encoding a and £ subunits of neuronal nAChRs. In the brain and peripheral ganglia these genes have distinct, yet overlapping patterns of expression. A number of neuronal nAChRs with distinct functional and pharmacological properties can be made in oocytes by pairwise combination of different a and fi subunits (Boulter, et al., 1987; Deneris et al., 1989;
Duvoisin et al., 1989; Papke et al., 1989; Bertrand et a l .,
1990; Boulter et a l ., 1990; Couturier et al ., 1990; Sawrok 28
et al., 1990; Luetje and Patrick, 1991).
Expression studies in Xenopus oocytes have shown that
the following combinations of subunits form functional
nicotinic receptors: a2/R2; a3 /R2 and a4/B2 (Boulter et
al., 1987; Wada et al., 1988). Different combinations of a
and R subunits form pharmacologically distinct neuronal
nAChR subtype, which posses varying affinities for agonists
and antagonists (Luetje and Patrick, 1991; Deneris et a l .,
1991). Antagonists such as hexamethonium, decamethonium and
tubocurarine and other cholinergic antagonists have been
shown to block receptor activation (Bertrand et a l ., 1990;
Couturier et al., 1990; Luetju et al., 1990; Luetje and
Patrick, 1991). However, the agonists, cytisine and 1,1 dimethyl-4-phenylpiperazinium (DMPP) and the antagonists,
Bgt 3.1 and neosurugatoxin are more potent agonists of neuronal nAChRs than muscle type nAChRs (Couturier et a l .,
1990; Bertrand et al., 1990; Luetju et al ., 1990; Luetje and
Patrick, 1991).
Neuronal nAChRs contain two ACh-binding sites (Whiting and Lindstrom, 1986; Lindstrom et al., 1987), like Torpedo and muscle nAChRs, and these sites interact in a positive cooperative manner. The subunits of neuronal nAChRs also associate to form a ligand gated ion channel (Lindstrom et al., 1987), and the electrophysiological properties of the channel are not much different from those of muscle AChRs
(Lipton et al ., 1987; Sakmann et al ., 1983). Like muscle 29
nAChRs, noncompetitive antagonists, also block neuronal
nAChR ion permeability (Ramoa et a l ., 1990; Rapier et a l .,
1987). Among the noncompetitive antagonists that also block
neuronal nAChRs are histrionicotoxin (Eldefrawi et al.,
1977), phencyclidine (Albuquerque 1980), MK-801
(Clineschmidt et al., 1982), hexamethonium and decamethonium
% (Bertrand et a l ., 1990), tubocurarine (Wada et al., 1988)
and neosurugatoxin (Luetje et al., 1990).
Neuronal nAChRs on Adrenal Chromaffin Cells
nAChRs on chromaffin cells resemble ganglionic nAChRs
of the autonomic nervous system since both are derived
embryologically from the neural crest. The unavailability of
suitable probes had prevented the identification and
biochemical characterization of nAChRs present in chromaffin
cells. a-Bgt, which blocks the function of Torpedo and muscle nAChRs (Fambrough, 1979), has no effect on neuronal nAChRs detected in PC12 cells, chromaffin cells, autonomic ganglia and the central nervous system (Kilpatrick et al.,
1981; Deneris et al., 1991).
Bgt 3.1 and mAb 35, two probes which have been useful in identifying neuronal type nAChRs, have been shown to recognize nAChRs on bovine adrenal chromaffin cells (Higgins and Berg, 1987). mAb 35 cross reacts with the functional nAChRs of chicken ciliary ganglion neurons (Halvorsen and
Berg, 1986) and bovine adrenal chromaffin cells (Higgins and Berg, 1987). This antibody binds to the main immunogenic region (a highly conserved determinant of unknown function which is very conformational dependent) of the muscle nAChR
(Ralston et a l ., 1987). Antibodies to the main immunogenic region of muscle and electric organ nAChR o subunit have been shown to induce clustering, internalization and degradation of muscle nAChRs (Merlie et al ., 1979).
Prolonged exposure to mAb 35 will specifically modulate chromaffin nAChR function and cause a decrease in the number of nAChRs (Higgins and Berg, 1988) . These observations provide evidence that the mAb 35 binding component on chromaffin cells is part of the nAChR.
The second probe, Bgt 3.1, has been shown to block function of neuronal nAChRs expressed in autonomic ganglia
(Jacob et al., 1984; Higgins and Berg, 1987) and in adrenal chromaffin cells (Higgins and Berg, 1987). Reports suggest that nAChRs on chromaffin cells are pharmacologically similar to other neuronal nAChRs (Higgins and Berg, 1987) .
They are activated by agonists such as nicotine, acetylcholine and carbachol and blocked by Bgt 3.1, hexamethonium, decamethonium, d-tubocurarine and other antagonists shown to block neuronal nAChRs (Higgins and
Berg, 1988b). It has been demonstrated that adrenal chromaffin nAChRs are desensitized by cholinergic agonists
(Clapman and Neher, 1984; Higgins and Berg, 1988b).
An a-Bgt binding site has been demonstrated on adrenal 31
chromaffin cells. Studies have indicated that the a-Bgt
binding component is distinct from the nAChRs on the cells,
since a-Bgt does not block receptor activation by agonists
(Kilpatrick, 1981; Higgins and Berg, 1988b).
Results obtained from binding and functional studies conducted to characterize adrenal chromaffin nAChRs suggest that adrenal nAChRs are indeed representatives of neuronal nAChRs (Higgins and Berg, 1988). These receptors like neuronal type nAChRs differ from muscle and electric organ nAChRs in both structure and regulation (Whiting and
Lindstrom, 1986; Halvorsen and Berg, 1987; Higgins and Berg,
1987, 1988).
RECEPTOR-CYTOSKELETON ASSOCIATIONS
Recognition of a stimulus at cell surfaces and transmission of signals from the external environment across the plasma membrane are key steps in activation of many cells. Association of surface receptors with the cytoskeleton is widely believed to be an early event resulting from cellular activation (Nicolson, 1976).
Numerous membrane receptors have been demonstrated to be associated with the cytoskeletal network, including receptors for nerve growth factor (Vale and Shooter, 1982) ,
PDGF (Zippel et al.,1989), transferrin (Hunt et a l ., 1989) and fibronectin (Horwitz et al., 1986). Association of these receptors with the cytoskeleton has been demonstrated 32
to play an important role in receptor function. Receptor-
cytoskeletal linkages are important for segregation of
receptors and ion channels in specific membrane regions
(Henis, 1989), signal transduction mechanisms (Carraway and
Carraway, 1989) and in receptor-mediated endocytosis
(Devreotes and Fambrough, 1975; Heinemann et a l ., 1977;
Higuchi et al., 1981).
Receptor-cytoskeletal associations have also been
demonstrated for muscle nAChRs. In postsynaptic regions of
both electric organ and muscle, nAChRs accumulate at very
high densities with little lateral diffusion in and out of
this area. nAChRs are transmembrane proteins that are
inherently capable of diffusion within the plane of the
membrane; therefore, these receptors must be anchored at the
synaptic site by interactions with other proteins. In adult muscle, the nACh are clustered in the postsynaptic membrane
of the neuromuscular junction. Prives et al. (1982)
demonstrated using cultured muscle cells that as these cells
age in culture the proportion of nAChRs forming clusters
increase and the receptors became more resistant to extraction with Triton, a treatment which leaves the
internal cytoskeleton essentially intact. Similarly, Stya and Axelrod (198 3a) have shown a direct correlation between the mobility of nAChRs in the plane of the membrane and their extractability by Triton. In these studies on cultured muscle cells, receptor clusters which were immobile 33
were also resistant to Triton-extraction, suggesting an
association with the cytoskeleton.
Various proteins at the synaptic site have been
implicated in being involved in anchoring nAChRs (Froehner,
1986). An intracellular 43 kD peripheral protein (known as
the 43K protein) has been shown to colocalize in equimolar
concentrations with nAChRs (LaRochelle and Froehner, 1986)
from electric organ (Sealock et a l., 1984) and skeletal muscle (Froehner et al., 1981). Recent studies by Froehner and coworkers (1990), using a Xenopus oocyte expression system, demonstrated that nAChRs are diffusely distributed when expressed in Xenopus oocytes; however, coexpression with the 43K protein results in clustering of nAChRs. In these studies, it was also shown that the 43K protein was capable of forming clusters when expressed in the absence of nAChRs. Similar results were obtained by Phillips and coworkers (1991), using a quail fibroblast cell line to express cDNAs for muscle nAChRs and the 43K protein. These researchers also observed that when nAChRs were expressed in the absence of the 43K protein, the receptors fail to cluster. However, when nAChRs were coexpressed with the 4 3K protein, clustering was observed on the cell surface.
Patches on the cell surface were also observed when the 43K protein was expressed alone. These observations together suggest that the 43K protein might participate in aggregation of nAChRs and possibly link the nAChRs to the 34
cytoskeleton to maintain their localization, as in
postsynaptic membranes (Froehner et al., 1990, Phillips et
al., 1991).
Various studies have provided evidence that
microtubules may participate in anchoring receptors. For
example, Connolly and Oldfin (1985) using cultured chick
muscle cells, have demonstrated that treatment of these
cells with a microtubule stabilizing drug, taxol, induces
aggregation of nAChRs. In other studies, Connolly (1984)
has shown that treatment of muscle cells with microtubule
disrupting drugs such as demecolcine and nocodazole, blocks
formation of nAChRs clusters. These results were consistent
with earlier studies by Stya and Axelrod (1983b), which
demonstrated that the microtubule disrupting drug,
colchicine, blocked formation of nAChR clusters on rat
myotubules. Therefore, these studies provide evidence that
microtubules might bind to nAChRs.
Interactions of membrane receptors with cytoskeletal
proteins are believed to play important roles in membrane
functions such as ion transport and signal reception
(Freedman, 1981). Young and Poo (1983), using Xenopus muscle cells, demonstrated that the time the nAChR associated ion channel remained open was longer for clustered than nonclustered receptors. These studies also imply that an association of nAChRs with the cytoskeleton is important for nAChR topography within the cell membrane and 35
for nAChRs channel function.
Similarly, studies have implicated a role for
microtubules in neuronal nAChRs. For example, it has been
shown that in adrenal chromaffin cells microtubule drugs
such as taxol, colchicine and vinblastine inhibit nicotine
mediated catecholamine release without affecting release
stimulated by other secretagogous (McKay and Schneider,
1984). Additional studies by McKay and Schneider (1985) have shown that taxol and vinblastine inhibit ACh induced
Ca+2 uptake in adrenal chromaffin cells without affecting K+ stimulated uptake. McKay and coworkers (1984) have also demonstrated that vinblastine interferes with histrionicotoxin binding to sites associated with the nAChR ion channel complex from Torpedo electric organ. These results suggest that these microtubule drugs are acting at the level of the nAChR to interfere with receptor function and binding. Furthermore, McKay (1988) has shown that taxol alters the microtubular network of adrenal chromaffin cells.
These studies together suggest that the action of the microtubule drugs are not on the terminal steps of exocytosis, but instead alter nAChR function by either a direct effect of the drugs on the receptor or an effect on microtubule links to the nAChRs of adrenal chromaffin cells.
Therefore, if indeed microtubules play a role in adrenal nAChR function, then other microtubule drugs should have the same or similar effects on the function of adrenal nAChRs and should affect adrenal microtubules under the same or similar conditions that affect receptor function. The hypothesis under investigation is that adrenal nAChRs are associated with microtubules and that this association may be important for receptor function. CHAPTER III
MATERIALS AND METHODS
MATERIALS.
Chemicals and Antibodies
DL-[7-3H(N)]norepinephrine (specific activity, 8-15
Ci/mmol), L-[N-methyl-3H]nicotine, mouse anti-tubulin antibodies, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulins and FITC-conjugated goat anti-rat immunoglobulins were obtained from DuPont New
England Nuclear Corporation, Boston, MA. The antibody, 6 -
11B-1, specific for acetylated tubulin, was a generous gift from Dr. Gianni Piperno (The Rockefeller University, N.Y.).
The cell line producing, the monoclonal antibody mAb3 5, was obtained from the American Type Culture Collection,
Rockville, MD. The following chemicals were obtained from the Sigma Chemical Company, St. Louis, MO: nicotine hydrogen tartrate, histamine hydrochloride, veratridine, podophyllotoxin, demecolcine (colcemid), nocodazole, poly-L- lysine hydrobromide (MW range 77,000 - 79,000), L-ascorbic acid, Trizma hydrochloride (Tris [hydroxymethyl] aminomethane hydrochloride), Trizma base (Tris
[hydroxymethyl] aminomethane base), dimethylsulfoxide (DMSO,
99 % pure). Tubulozole hydrochloride was obtained from
37 38
Research Biochemicals Incorporated, Natick, MA. Vinblastine
and vincristine were gifts from the Eli Lilly Company,
Indianapolis, IN. Taxol was obtained from the National
Products Branch, Division of Cancer Treatment, National
Cancer Institute (Bethesda, MD) and was dissolved in DMSO.
ScintiVerse E liquid scintillation cocktail was acquired
\ from Fisher Scientific, Cincinnati, OH. All other reagents used were of analytical grade.
Cell Culture Supplies
Dulbecco's Phosphate Buffered Saline (D-PBS) 10X, powdered Dulbecco's Modified Eagle Medium (DMEM, low glucose and high glucose) and Protein Free Hybridoma Media (PFHM-II) were obtained from Gibco-BRL Laboratories , Grand Island,
NY. The following chemicals were obtained from the Sigma
Chemical Company, St. Louis, MO: antibiotic/antimycotic
100X solution (amphotericin B, 25 /ig/ml; penicillin, 10,000
U/ml; streptomycin, 5,000 /xg/ml), L-glutamine, gentamicin sulfate, Percoll, 5-flurodeoxyuridine (FDU), cytosine arabino-furanoside (Ara-C), HEPES (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]), fetal calf serum
(FCS, heat inactivated) and deoxyribonuclease (DNase, Type
IV) and bovine serum albumin, fraction V powder (BSA).
Collagenase Type I, (activity 196 - 210 U/mg) was acquired from Worthington Biochemical Corporation, Freehold, NJ.
Sterivex-GS filter units (0.22 jum) were obtained from 39
Millipore Corporation, Medford, MA. Disposable syringe
filters (0.22 /xm) were obtained from PGC Scientifics Co.,
Gaithersburg, MD. Falcon Primaria tissue culture plates (6-
well plates, 35mm), Corning 24 well (16 mm well) and 12 well
(25 mm well) plates, culture flasks (T25, T75 and T150 cm2)
and Spectra mesh (60, 105 and 210 /xm) were obtained from
Fisher Scientific, Cincinnati, OH.
METHODS
Adrenal Chromaffin Cell Isolation
Adrenal chromaffin cells were isolated from bovine
adrenal glands obtained immediately after slaughter from
Herman Falter Packing Company, Columbus, OH, and used within 30 min of their removal. The glands were kept on ice during transport to the laboratory. Adrenal chromaffin cells were isolated from the intact gland under sterile conditions as described by McKay and Schneider (1984).
After removing all the fatty tissue, 1 mm incisions were made over the entire surface of the gland to facilitate perfusion. A 3 ml syringe was used to gently inject 3 mis of sterile, Ca+2-free, Locke's solution (containing: 0.14 M
NaCl, 5.4 mM KC1, 1.0 mM NaH2P04, 11 mM glucose, 250 ng/ml amphotericin B, 100 U/ml penicillin and 50 /xg/ml streptomycin, 15 mM HEPES, pH 7.3 - 7.5) gently injected into the central vein of the gland. This procedure was repeated three times, to aid in the removal of residual 40
peristaltic pump was set up to perfuse the glands with
Locke’s solution. The tubing to be used in the perfusion
was kept in 70 % ethanol overnight, then rinsed with 50 ml
of Locke's solution prior to use. One end of the tubing was
inserted into the adrenal vein and the other end was
immersed in Locke's solution. Three to four glands were then placed on a gauze covered, wire mesh on a ring stand, suspended over a 1000 ml beaker (Fig. 3A). The glands were covered with a piece of gauze and moistened with Locke's solution. Glands were then perfused through the adrenal vein with 500 mis of Locke's solution at room temperature.
Following the perfusion with Locke's solution, a 3 ml syringe was used to gently inject 3 mis of sterile Locke's solution containing 0.05 % collagenase, into the central vein of the gland. This procedure was repeated one more time to aid in the digestion of the medulla. The glands were then placed into a 100 ml beaker containing enough 0.05
% cold collagenase solution to cover the glands. The tubing was removed from the Locke's solution and placed into the
100 ml beaker. The glands were perfused with the collagenase solution for 20 to 3 0 min (Levitt, 1984; McKay and Schneider, 1984).
After the collagenase perfusion, the glands were Figure 3. A. Set up of the perfussion apparatus. B. Cross- section of the adrenal gland, exposing the digested adrenal medulla (arrow).
41 42
W Buchlet 43
Figure 3 continued 44
removed from the collagenase solution and an incision made
around the perimeter of each gland. The medulla was
carefully separated from the cortex by a peeling motion.
When all the medulla was separated from the cortex (Fig.
3B), it was placed in a mincing bowl containing a small
amount of cold Locke's solution.
Following separation of all medullae, the tissues were minced (1-2 mm2) with 2 small scissors for several minutes. The minced tissue was then transferred to a 50 ml sterile centrifuge tube, 20 - 25 mis of collagenase solution added and the tissue incubated for 60 min at 3 7°C in a shaking waterbath (Precision Dubnoff Metabolic Shaking
Incubator, setting 4.5). After the digestion period, the tissue was separated from the collagenase solution by filtration through 250 jum nylon mesh. The filtrate containing dissociated cells was collected in a sterile 50 ml centrifuge tube. Cold Locke's solution was added to the tube (through the nylon mesh) to bring the final volume to
50 ml. The tube was then centrifuged at 800 rpm (75 xg) for
10 min in a bench top centrifuge (IEC-HN-SII Centrifuge.
The supernatant was carefully aspirated and discarded, and the cell pellet was gently resuspended in 10 ml of Locke's solution and stored on ice. The undigested tissue remaining on the nylon filter was placed in a sterile 50 ml centrifuge tube, 20 - 25 ml of fresh collagenase solution added and the digestion, filtration, and centrifugation processes were 45 repeated, as described above.
The cell suspensions from the first and second
digestions were pooled in a 50 ml centrifuge tube. Locke's
solution supplemented with 0.5 % BSA was added to bring the
final volume to approximately 50 ml. The cell suspension
was centrifuged at 800 rpm (75 xg) for 10 min. The cell
pellet was gently resuspended in 10 mis of Locke's solution
with BSA and filtered through a 100 /xm nylon mesh. The cell
filtrate was collected in a sterile 50 ml centrifuge tube.
Cold Locke's solution with BSA was added to the tube
(through the nylon mesh) to bring the final volume to 39
mis. The cell suspension was added to Percoll (pH adjusted
to approximately 7.2 - 7.4 prior to use), and this cell
suspension was centrifuged at 17,500 rpm (38,000 xg) in a
Sorvell refrigerated centrifuge (Sorvell RC-5B refrigerated
superspeed centrifuge, Dupont, N.Y.) at 4°C for 40 min. The utilization of this Percoll gradient technique to separate live from dead cells greatly enhanced the viability of the final cell preparation. Following centrifugation in the
Percoll gradient, the upper layer of dead cells was aspirated and the remaining viable cells were resuspended in
Locke's with 0.5 % BSA. The cells were then centrifuged at
1000 rpm (110 xg) for 15 min. After this centrifugation, the supernatant was discarded and the cells resuspended in a given volume of Locke's with 0.5 % BSA. The number of cells in suspension and their viability were determined using a 46
vital dye exclusion test with 0.4 % erythrosin B. Live
cells with intact membranes excluded the dye while dead
cells appeared red from an accumulation of erythrosin B dye.
The number of cells was determined using a Bright-Line
Hemocytometer, Reichert Scientific Instruments, Buffalo, NY.
Cell viability was generally 88 - 97 %. The cell suspension
was then brought to a final Volume of 50 ml with DMEM
supplemented with 10 % FCS, glutamine (200 juM), penicillin
(100 U/ml), streptomycin (50 /xg/ml), amphotericin B (0.25
Mg/ml) , 5-fluorodeoxyuridine (10 /liM) , gentamicin sulfate (40
n g / m l ), and centrifuged at 800 rpm (75 xg) for 10 min. The
cell pellet was gently resuspended with 8 ml of supplemented
DMEM and filtered through 60 nm nylon mesh. The filtrate
was collected in a sterile 150 ml beaker. The cell
suspension was then diluted to 1 x 106 cells/ml with
supplemented DMEM.
Cells to be used in secretion expriments were plated in
24-well culture plates containing supplemented DMEM culture media (1 ml/well). These isolated chromaffin cells were plated at a density of 1 - 2 x 10s cells/well using an
Eppendorf Repeat Pipetter. Cells to be used for immunocytochemistry were plated in 6-well (35 mm, 3 ml supplemented DMEM/well) culture plates containing glass coverslips (18 x 18 mm) at a density of 1 - 2 x 105 cells/well. Cells to be used for binding experiments were plated in 12-well (25 mm, 1.5 ml supplemented DMEM/well) culture plates at a density of 1 x 106 cells/well . After
plating the chromaffin cells, the plates were placed in a
humidified, 37°C, 5 % C02 incubator (Precision Scientific,
Chicago, IL).
Catecholamine Secretion Studies.
Loading Cells with [3H]Norepinephrine
In order to measure catecholamine secretion from
cultured bovine adrenal chromaffin cells, the cells were
first loaded with [3H]norepinephrine. This radiolabelled
catecholamine is taken up into the chromaffin cells by a
neuronal uptake I-like mechanism (McKay, 1989) and stored in
granules prior to release from the cells. The general
procedure for loading the cells with [3H]norepinephrine are
described by McKay and Schneider (1984). Modifications of
this method which were necessary for some of the procedures
found in this work will be outlined below.
The culture medium was aspirated from the 24-well
culture plates and the cells washed once with 1 ml of a
physiological salt solution (PSS, containing 140 mM NaCl,
4.4 mM KCl, 1.2 mM KH2P04, 3.6 mM NaHC03, 1.2 mM MgS04, 2 mM
CaCl2, 10 mM glucose, 5 mM HEPES and 0.5 % BSA, pH 7.3 -
7.35). After washing the cells, 200 til of loading buffer was added to each well. The loading buffer was prepared by adding ascorbic acid (0.01 %) and [3H]norepinephrine (0.1 48
/iM) to PSS. The plates were then placed in a humidified,
37°C incubator for 1 - 2 hr. After the incubation period,
the cells were washed three times (15 min each) with l ml of
PSS to remove any unincorporated radioactivity .
Catecholamine Secretion Experiments
Catecholamine secretion experiments' were performed in
PSS which contained 2 mM Ca+2 and 0.5 % BSA. In experiments
where chromaffin cells were stimulated with 56 mM KC1, the
amount of Na+ in the buffer was decreased to maintain
isotonicity. Similarly, when BaCl2 was used to stimulate
cells, the buffer composition was changed to Ba+2' 2 mM;
NaCl, 140 mM; KC1, 5.6; MgCl2, 1.2 mM; glucose, 10 mM;
HEPES, 5mM (pH 7.2-7.4); and 0.5 % BSA. Throughout these
studies, triplicate wells were used for each treatment
group.
In experiments in which the effects of microtubule
drugs on nicotine-stimulated catecholamine release were
investigated, the cells were pretreated at room temperature with these compounds for 3 0 min prior to stimulation which occurred in the continued presence of the drug. For time course studies, cultured bovine adrenal chromaffin cells were incubated at room temperature for different time periods (0, 5, 10, 30, 60 and 180 min) with the microtubule drug at a concentration approximating IC80 values. In studies in which the reversibilty of the effects of microtubule drugs was examined, the cells were pretreated
with approximate IC80 concentrations of the agent for 1 hr.
After the treatment period, the drug solution was removed
and the cells were washed for different time periods (120,
60, 30, 10, 2 and 0 min). After the washes, the cells were
stimulated with nicotine (10 fxM) in the absence of the microtubule drug, except for the group of cells where the
drug was not removed (0 min). Basal (PSS alone) and control
nicotine-stimulated (10 nM nicotine in PSS) cells were preincubated with PSS for 3 0 min during the pretreatment period. Cells used to measure basal release were also kept in PSS during the 10 min stimulation period.
In studies where the selectivity of mAb 3 5 for nAChRs was tested, cells were incubated with 10 and 100 nM mAb 3 5 for 20 hrs. At the end of the incubation period, the cells were loaded with [3H]norepinephrine, in the presence of either 10 nM or 100 nM mAb35, for 1 hr. After the loading period, the cells were washed three times (15 min each) with
1 ml of PSS in the presence of mAb 3 5 (cells were exposed to mAb 35 for a total of 24 hr). The cells were then stimulated with either nicotine (10 m M) or 56 mM K+, in the absence of mAb 35.
For all secretion experiments, after the stimulation period, the medium from each well was transferred to 7 ml glass vials containing 5 ml scintillation clocktail, mixed well and counted by liquid scintillation spectroscopy. The 50
radioactivity remaining in the cells was determined by
extracting the cells with 8 % (w/v) TCA (0.5 ml per well)
for 15 min. The cell extract was placed in glass vials,
mixed with 5 ml of scintillation cocktail and counted for 10
min by liquid scintillation spectroscopy. The sum of the
secreted and the TCA-extractable radioactivity represented
total incorporated [3H]norepinephrine. Results were
expressed either: 1) as the fraction of the total
incorporated [3H]norepinephrine released under the treatment
conditions (i.e., secreted [3H]norepinephrine divided by
total incorporated [3H]NE) or 2) as % control (i.e. the
fractional amount released under each treatment condition divided by the fraction released by secretagogue).
Tubulin Immunocytochemistry
General Procedure
An indirect immunofluorescence technique was used to investigate drug effects directly on the adrenal microtubular network, as described by McKay (1989). DMEM was aspirated, and the cells in each well of a 6-well plate were pretreated with the drugs to be tested. After treatment, the coverslips (with attached cells) were removed from the 6-well plate and labeled on the lower right hand corner with a diamond pen. The coverslips were then washed one time by placing into a Columbia jar containing
Dulbecco's phosphate buffered saline (D-PBS) for 5 min. Following the wash, the D-PBS was aspirated off and
enough 3.7 % formaldehyde solution added to the coverslips
in the Columbia jar to cover them. The cells were incubated
in the fixing solution for 30 min. After fixation, the
formaldehyde solution was aspirated from the Columbia jar
and a 0.2 % Triton X-100 in 3.7 % formaldehyde solution
added. The cells were extracted for 10 min at room
temperature. At the end of the extraction period the
coverslips were placed on a coverslip rack and washed
extensively by dipping the rack 15 - 20 times into a 150 ml
beaker containg fresh D-PBS solution. The coverslips were
then placed back into a clean Columbia jar with fresh D-PBS and incubated for 10 min. The cells were then incubated in
50 % acetone for 3 min, followed by a 5 min incubation in
100 % acetone and once again incubated in 50 % acetone for 3 min. At the end of the incubation period, the acetone was aspirated, the cells rinsed twice in D-PBS, and then incubated for 10 min in D-PBS.
Following the wash, each coverslip was individually removed from D-PBS and any excess buffer removed by tilting the coverslip and blotting the edges with a tissue. Mouse anti-tubulin antibody (1/10 - 1/20 dilution) was added directly to the cells, approximately 20 - 40 /xl, making sure that the entire coverslip was covered with the antibody solution. The coverslip was then placed on top of the 6- well plate. The same procedure was repeated for the other 52
coverslips. The coverslips were then placed in a moist
chamber and incubated for 60 min.
After incubation with the primary antibody, the
coverslips were removed from the moist chamber and each
coverslip carefully rinsed with D-PBS using a squirt bottle
to wash off excess antibody. The coverslips were then
washed extensively by placing into the coverslip rack,
dipped 15-20 times into a 150 ml beaker containing fresh
D-PBS twice, placed back into a Columbia jar with fresh D-
PBS and incubated for 10 min in fresh D-PBS. Following the
wash, each coverslip was individually removed from D-PBS and
any excess buffer removed by tilting the coverslip and
blotting the edges with a tissue. FITC-conjugated goat
anti-mouse immunoglobulins (1/150 - 1/200 dilution) was
added directly to the cells, approximately 20 - 40 yul,
making sure that the entire coverslip was covered with the
antibody solution. Each coverslip was then placed on the
cover of a 6-well plate, then placed in a moist chamber and
incubated for 60 min.
At the end of the incubation period with the FITC-
labeled secondary antibody, the cells were extensively washed as described above. After the 10 min wash in D-PBS, the coverslips were removed from the Columbia jar with forceps, the edges blotted dry with a tissue and wet mounted
(cells facing down) onto a 10 pi drop of mounting media
(9:1, glycerol:D-PBS). Clear nail polish was applied around 53
the edges of the coverslip to seal the edges. The specimens
were examined with a Nikon Labophot microscope equipped with
epifluorescent optics and photographed using Tri-X-Pan 400
ASA Kodak black and white print film.
Acetylated Tubulin Studies
The indirect immunofluorescence procedure used in these
studies was identical to that described above with a few
minor changes. After fixing, extracting and incubating the
cells in acetone, the cells were washed twice in D-PBS
containing 1 % BSA for 10 min, to decrease the amount of
nonspecific antibody binding. The cells were then incubated
in D-PBS containing BSA for 3 0 min. The anti-acetylated
tubulin antibody (6-11B-1) was then added directly to the
coverslips and incubated as described for the anti-tubulin
antibody above. The rest of the procedure is as described above.
[3H]Nicotine Binding Studies
Rat Brain Membrane Preparation.
Membranes were prepared from Sprague-Dawley rat brain tissue, according to the method of Romano and Goldstein
(1980). Briefly, whole brains were removed, rinsed with ice-cold buffer containing Na2HP04, 8mM; KH2P04, 1.5 mM;
KC1, 3 mM; NaCl, 120 mM; disodium ethylenediamine- tetraacetate (EDTA), 2mM; HEPES, 20 mM (pH 7.4); 5 mM 54
iodoacetamide; 0.1 mM phenylmethylsulfonyl fluoride (PMSF),
several times to remove any blood and homogenized. The
homogenate was centrifuged (25000 xg, 0°C) for 20 min. The
pellet was resuspended in distilled, deionized water and
kept on ice for 60 min before centrifugation as described
above. The pellet was then resuspended in the assay buffer
to a final concentration of 1 mg/ml. The assay buffer was
the same as the buffer used to prepare the membranes with
the addition of 1 mM MgCl2 and 2 mM CaCl2 and the
elimination of EDTA and PMSF. The protein concentration was
then determined by the Bradford method (Bradford, 1976)
using a commercial Bradford reagent (Bio-Rad Loaboratories,
Cambridge, MA). Bovine serum albumin (BSA) was used as the
protein standard.
[3H]Nicotine Binding to Rat Brain Membrane Fragments.
To determine the effects of microtubule drugs on
[3H]nicotine binding, binding assays were performed using the procedure of Lippiello and Fernandez (1982) with slight modification. Membranes (450 fig protein) were incubated with [3H]nicotine (15 nM) and 1 /iM a-Bgt in the absence or presence of the microtubule drugs in a total volume of 500
/xl at room temperature for 60 min. Incubations were terminated by adding approximately 4 ml of ice-cold assay buffer (described above) and rapidly filtering the mixture through Whatman GF/B filters, using a Brandel binding 55
manifold (Brandel Research and Development Laboratories,
Inc., Gaithersburg, MD). The filters were soaked in 0.1 %
polylysine for over 3 hr at 5°C to reduce nonspecific
binding (Romano and Goldstein, 1980). After the initial
filtration of the assay mixture, filters were continuously
washed with ice-cold assay buffer for 15 sec. The filters
were then placed in counting vials and mixed vigorously with
7 ml of ScintiVerse E. Samples were counted in a Beckman LS
6800 liquid scintillation counter at 30 - 40 % efficiency.
Nonspecific binding was determined in the presence of excess
carbachol (1 mM).
[3H]Nicotine Binding to Intact Adrenal Chromaffin Cells
After isolation, chromaffin cells that were going to be
used for whole cell [3H]nicotine binding studies were plated
onto Corning 12 well (25 mm) plates, containing DMEM, (1.5
ml/well) , supplemented with 10 % (v/v) FCS and 200 /zM, at a
density of 1x10s cells/well. After plating the culture
plates were placed in a humidified 37°C, 95 %/5 % C02
incubator. Cultures were used for experiments 3 - 5 days
after plating.
[3H]Nicotine binding to intact adrenal chromaffin cells
was determined as described by Higgins and Berg (1988) .
Culture plates were removed from the incubator, the culture medium aspirated from the wells and the cells washed once with 1 ml of DMEM. The culture media was then aspirated and replaced with 500 jul of DMEM containing 10 nM [3H]nicotine
and 1 jliM a-Bgt. The cells were then placed in a humidified,
37°C incubator for 70 min. At the end of the incubation
period the media was aspirated using a Millipore vacuum
pressure pump, Bedford, MA., (vacuum set at 22 in of
mercury) and the cells quickly washed 4 times (<10 sec,
total wash time) with 1 ml of rinse buffer (pH 7.4, 5 mM
HEPES, 137 mM NaCl, 5.4 mM KC1, 0.8 mM MgS04, 0.9 mM
Na2HP04, 0.4 mM KH2P04, 1.8 mM CaCl2, 5.6 mM glucose and 2
mg/ml BSA) at 37°C. This procedure was repeated for the
remaining wells. Cells were then dissolved by incubating
the cells in 500 (il of a 1 M NaOH solution for 60 min. At
the end of the incubation period 100 /xl aliquots from each
well were added to 5 ml polystyrene plastic test tubes and
assayed for protein by the method of Lowry et al (1951)
utilizing BSA as the standard. The remaining 400 nl aliquot
of lysate was neutralized with 4 M HCL, mixed with
scintillation cocktail and analyzed by liquid scintillation
counting using a Beckman 6800 liquid scintillation counter.
[125I]mAb 35 Studies
Growth and Maintenance of mAb 35 Secreting Hybridoma Cell Line
The hybridoma cell line secreting mAb 35, a monoclonal antibody reactive with nAChRs, was obtained from ATCC. Upon arrival, the cell suspension containing 3 x 106 cells/ml was added to a T25 flask and diluted (1:25) in DMEM (high 57
glucose) containing 10 % fetal calf serum, 2 mM glutamine,
50 pig/ml gentamycin, penicillin (100 U/ml), streptomycin (50
Mg/ml), and amphotericin B (0.25 nq/ m l ), as directed in the
product sheet.
Hybridoma cells were maintained in culture as suggested
by Harlow and Lane (1988). The hybridomas were examined
daily under a microscope for bacterial or 'fungal growth and
for cell growth. Every 2 - 3 days or when the cell density
appeared high enough Protein Free Hybridoma Media (PFHM-II)
was added to the T25 flask to maintain a cell density
between 1 x 105 and 1 x 106. This process allowed gradual dilution of the DMEM with PFHM-II. When the cell density was high and the culture media reached the neck of the T flask, the hybridomas were split by taking aliquots of the cell suspension and transferring it to a larger T flask (T75 or T100). PFHM-II was added to maintaine the cell density between 1 x 10s and 1 x 106 cells. This procedure of diluting or splitting the hybridoma cells was repeated until a desired amount of supernatant containing mAb 35 was obtained.
Purification of mAb 35
mAb 3 5 was purified by ammonium sulfate precipitation from hybridoma culture media as described by Harlow and Lane
(1988). Briefly, 100 ml of tissue culture supernatant were centrifuged at 5000 rpm (3000 xg) for 30 min in a 58
refrigerated centrifuge (Sorvall, RC-5B, refrigerated
superspeed centrifuge, Dupont, N.Y.). The supernatant was
transferred to a clean 400 ml beaker with a stir bar and
placed on a magnetic stirrer. While the antibody solution
stirred gently, 66.7 mis of saturated ammonium sulfate (761
g/L, pH 7.3 - 7.4 with NH40H) were slowly added to bring the
final concentration to 40 % saturation. The beaker with the
antibody-containing solution was then placed in a
refrigerator at 4°C and left covered overnight.
The ammonium sulfate precipitated antibody was
centrifuged at 5000 rpm (3000 xg) for 30 min in a Sorvell
refrigerated centrifuge, to remove the precipitated antibody
from the media. After centrifugation, the supernatant was
discarded and the pellet was resuspended in 0.1 volume (10
mis) of the starting supernatant in D-PBS (pH 7.2). The
antibody solution was then transferred to a 2 0 cm dialysis
tubing (Spectra/Por membrane, MWCO 50,000, Fisher Scientific
Co., Cinncinati, OH), placed in 2 L of D-PBS containing 0.02
% sodium azide and dialyzed against 2 changes of D-PBS
overnight at 4°C. The antibody solution was then removed
from the tubing and placed in a centrifuge tube and spun at
5000 rpm (3000 xg) for 30 min in a Sorvall refrigerated
centrifuge, to remove any remaining debris. The
concentration of the antibody was then determined by the
Bradford method (Bradford, 1976) using a commercial Bradford reagent (Bio-Rad Loaboratories, Cambridge, MA). Immunoglobulin (IgG) was used as the standard. The protein
concentration was usually 1 - 2 mg/ml. The antibody
solution was then aliquoted and stored below 0°C.
Iodination of mAb 35
An aliquote (333 /il) of purified mAb 35 (1.5 mg/ml) was
radioiodinated to a specific activity of 44 /xCi/ml (or 22
Ci/mm) using the chloramine T technique as described by
Lindstrom et a l . (1981) for iodination of a-Bgt. This
procedure was performed by Dr. Michaelle V. Darby, at The
Ohio State University Comprehensive Cancer Center Radiation
Laboratory.
l125I]mAb 35 Binding to Intact Adrenal Chromaffin Cells
[125I]mAb 35 binding to intact adrenal chromaffin cells was determined as described by Higgins and Berg (1987).
Culture plates were removed from the incubator, the culture medium aspirated from the wells and replaced with 500 /il of
DMEM containing 5 nM 125ImAb 35. The cells were then placed in a humidified 37°C, 5 % C02 incubator for 60 min. At the end of the incubation period the media was aspirated using a vacuum pump (Millipore vacuum pressure pump, set at 22 inches of Hg). The cells were quickly washed (<10 sec) 4 times with 1 ml of rinse buffer containing 137 mM NaCl, 5.4 mM KC1, 0.8 mM MgS04, 0.9 mM Na2HP04, 0.4 mM KH2P04, 1.8 mM
CaCl2, 5.6 mM glucose, 5 mM HEPES (pH 7.4) and 2 mg/ml BSA at 3 7°C. This procedure was repeated for the remaining
wells. Cultures were then dissolved in 500 /il of a 1 M NaOH
solution for 60 min. At the end of the 60 min the cells
were scraped in the NaOH solution with a rubber policeman to
remove any residual cells remaining attached to the plate.
The dissolved cells were then added to 7 ml glass vials and
analyzed for 125I with a Beckman Model 8000 gamma counter.
Nonspecific binding, determined by adding 0.5 /iM of
unlabeled antibody to the incubation media, usually
constituted 14 - 26 % of the total binding and was
subtracted to yield specific binding.
[125I]mAb 35 Binding to Triton-Extracted Adrenal Chromaffin Cells
After labelling the cultures with [125I]mAb 35, cells
were Triton-extracted as described by Prives et al. (1982).
Briefly, the labelled cells were washed once with a 1 ml
volume of Buffer A, pH 7.4, containing 0.3M sucrose, 50 mM
NaCl, 1 mM MgCl2 and 10 mM HEPES. Buffer A was then
aspirated and the cells extracted for 5 min with 500 /il of
Buffer A containing 0.5 % Triton X-100 at room temperature.
After the extraction period, the Triton buffer containing the Triton-soluble cell components was removed from the cells and transferred to 7 ml glass vials. The radioactivity remaining on the wells was determined by dissolving the Triton-insoluble cell fraction with 500 /il of a 1 M NaOH for 1 hr and scraped in NaOH. The dissolved 61
Triton-insoluble cell components were then added to 7 ml
glass vials and analyzed for 125I with a Beckman Model 8000
gamma counter.
In experiments in which the effect of microtubule drugs
on [125I]mAb 3 5 binding to Triton-extracted cells were
examined, [125I]mAb 35 binding was performed in the presence
or absence of the microtubule drugs. [125I]mAb 3 5 binding
was performed as mentioned above. After binding of
[125I]mAb 35, the cells were extracted and the amount of
radioactivity associated with the Triton-insoluble cell
component determined using a gamma counter, as described
above.
Calculations and Statistics
Secretion data are presented as arithmetic means ± SEM
except for IC50 values which are presented as geometric means (95 % confidence limits); n represents the number of observations carried out in triplicate. Inhibition curves were generated by a nonlinear least squares fit for a general sigmoid curve (InPlot 3.1, GraphPad, San Diego,
CA.). Data were analyzed by Dunnett's Test with significance at the p < 0.05 level. The data acquired in binding studies are presented as arithmetic means ± SEM; n represents the number of observations carried out in duplicates or triplicate. Data were analyzed by 2 Way ANOVA
Test with significance at the p < 0.05 level. CHAPTER IV
RESULTS
Effects of microtubule drugs on nicotine-stimulated [3H]norepinephrine release from adrenal chromaffin cells \ Drugs known to alter microtubule function, such as taxol and vinblastine, have been shown to inhibit nicotine- stimulated catecholamine release (McKay and Schneider, 1984;
McKay et a l ., 1985). In order to further characterize the effects of other microtubule drugs on nAChR-stimulated catecholamine release in cultured bovine adrenal chromaffin cells, the concentration-response effects of other microtubule drugs on nAChR-stimulated catecholamine release were examined. Vincristine, tubulozole, podophyllotoxin and demecolcine inhibited nicotinic receptor-stimulated release of [3H]norepinephrine in a concentration-dependent manner
(Fig. 4) with IC50 values of 3(1-10), 5(2-10), 8 (4-15) and
19 (9-39) juM, respectively (Table 1). Catecholamine release stimulated by 10 jttM nicotine was almost completely abolished by all four compounds at a concentration of 100 mM.
Effects of microtubule drugs on catecholamine secretion mediated by various noncholinergic secretagogues
McKay and coworkers (1984, 1985) have previously
62 63
Table 1. Comparison of IC50 values of various microtubule drugs for inhibition of adrenal nicotine-stimulated catecholamine release and for inhibition of in vitro tubulin polymerization.
Catecholamine Tubulin Drug Release Polymerization IC50 (/iM)a IC50 (MM)
Vincristine 2.8 (0.8-10) 0.1b, 2.0C
Tubulozole 4.6 (2-10) 0.3d, 4.0e
Podophyllotoxin 8.1 (4.4-14.7) 0.5f, 1.0g
Demecolcine 19 (9.3-39.4) NKh
aValues were derived from concentration-response curves. bHimes et a l ., 1977 cOwellen et a l ., 1972 (porcine brain tubulin) dVan Ginckel et a l .,1984 (rat brain) eDuanwu et a l .,1989 (L1210 murine leukemia tumor cell line) fJordan et a l .,1983 gHoebeke et a l ., 1975 hNot Known 64
CO
cr
o q : c_ m -M CL oC o • DEHECOLCINE
■ P0D0PHYLL0T0XIN oq : ▲ TUBULOZOLE ♦ VINCRISTINE co
[DRUG] (M)
FIGURE 4. The concentration-response effects of several microtubule drugs on nAChR-stimulated adrenal catecholamine release. Cultured adrenal chromaffin cells were pretreated for 30 min with increasing concentrations of vincristine (filled diamonds), tubulozole (filled triangles), podophyllotoxin (filled squares) or demecolcine (filled circles) prior to stimulation with 10 nM nicotine in the continued presence of the drug. Results are expressed as a percentage of the nicotine-stimulated control response. Basal [3H]norepinephrine fractional release was 0.057 ± 0.005 and nicotine stimulated fractional release was 0.207 ± 0.014. Values represent means ± SEM of 8 to 10 experiments performed in triplicate. 65
demonstrated that the inhibitory effects of taxol and
vinblastine on adrenal catecholamine secretion are selective
for release stimulated through activation of nAChRs. To
determine whether other microtubule drugs show similar
selectivity, the effects of tubulozole, demecolcine,
podophyllotoxin and taxol on adrenal catecholamine release
stimulated by noncholinergic secretagogues (Ba+2, K+,
veratridine and histamine) were investigated. Tubulozole
(10 M) , demecolcine (100 /liM) , podophyllotoxin (100 /iM) and
taxol (10 /iM) significantly inhibited nicotine-stimulated
[3H]norepinephrine release by 63 %, 82 %, 76 % and 61 %,
respectively (Table 2). However, at these same
concentrations the microtubule drugs did not affect release
stimulated by 56 mM KC1, 2 mM Ba+2 or 50 /zM veratridine.
The ability of these drugs to interfere with histamine-
mediated [3H]norepinephrine release was also investigated to
determine if the effect of the microtubule drugs was
specific for release stimulated by receptor mechanisms.
None of the microtubule drugs significantly altered
histamine-stimulated catecholamine release (Fig. 5). Basal
release of [3H]norepinephrine was not significantly affected
by any of the microtubule drugs tested (Table 3).
Effects of microtubule drug treatment time on nicotine- stimulated catecholamine secretion
Since it appears that microtubule drugs only inhibit catecholamine release mediated by nAChR in cultured bovine Table 2. Selective inhibition of nAChR-stimulated adrenal catecholamine release by microtubule drugs.a
Catecholamine Release (fractional)
10 fiM 56 mM 2 mM 50 flM Nicotine KCl Barium Veratridine
Basal (nonstimulated) 0.059±0.011 0.041±0.008 0.07110.016 0.03610.007
Secretagogue 0.165±0.016 0.122±0.016 0.41710.048 0.07410.010
+ Taxol, 10/jM 0.100±0.011c 0.12510.023 0.39310.037 NDb
+ Tubulozole, 10/iM 0.098±0.010c 0.08710.013 0.32810.020 0.04910.047
+ Demecolcine, 100/xM 0.078±0.013c 0.10110.015 0.41610.043 0.07210.010
+ Podophyllotoxin lOOfiM 0.085±0.016° 0.09410.017 0.39010.031 0.05510.008
aCultured adrenal chromaffin cells were pretreated for 30 min with the drug prior to stimulation in the continued presence of the drug. Results are expressed as a fraction of the total incorporated [3H]norepinephrine release during the 10 min stimulation period. Values represent means ± SEM of 5 to 7 experiments. “Not determined cSignificantly different from the corresponding secretagogue-stimulated controls (p < 0.05). 67
Table 3. Effects of microtubule drugs on basal (nonstimulated) catecholamine release.3
Basal Catecholamine Release3
Basal (control) 0.022 + 0.004
+ Demecolcine (100 ptM) 0.028 + 0.005
+ Podophyllotoxin (100 /iM) 0.032 + 0.003
+ Taxol (10 fJLM) 0.045 + 0. 004
+ Tubulozole (10 tiM) 0.045 + 0. 008
+ Vincristine (10 /iM) 0.032 + 0.003 aCultured adrenal chromaffin cells were treated for 30 min in the absence (control) and presence of the indicated microtubule drug. The amount of [3H]norepinephrine was measured during a 10 min period. Results are expressed as a fraction of the total incorporated [3H]norepinephrine released. Values represent means ± SEM of 5 to 7 experiments. None of the treatments were statistically different from control (p < 0.05). 68
|SECfETA60GUE Q tAX (10 |N) How (ioo 0 ) @ t u b (10 *) 0 N O C (100 |N) 0 V C R (10 IN) 0 P O O (100 IN)
NICOTINE (10 mM) HISTAMINE (500 mM)
FIGURE 5. Effects of various microtubule drugs on [3H]norepinephrine release stimulated by 10 /xM nicotine or 500 /xM histamine in cultured bovine adrenal chromaffin cells. Cells were pretreated for 30 min with 100 /xM demecolcine (DEM) , 100 /xM podophyllotoxin (POD) , 100 /xM nocodozole (NOC) , 10 /xM taxol (TAX) , 10 /xM tubulozole (TUB) or 10 fM vincristine (VCR) prior to stimulation with agonist in the continued presence of the drug. Results are expressed as a percentage of the respective secretagogue- stimulated control response. Basal [3H]norepinephrine fractional releases were 0.059 ± 0.011 and 0.045 ± 0.005 for nicotine and histamine treatment groups, respectively; stimulated fractional releases were 0.224 ± 0.02 and 0.206 ± 0.011, respectively. Results significantly different from control are marked by an asterisk (p < 0.05). Values represent means ± SEM of 6 to 10 experiments performed in triplicate. 69
adrenal chromaffin cells, it was of interest to further
characterize the manner by which these compounds elicit this
effect. One way of characterizing this effect is by
examining the length of time required for the microtubule
agents to inhibit nAChR-mediated [3H]norepinephrine release.
Cells were pretreated for various time periods (0, 5, 10,
30, 60, 12 0 min) with concentrations of microtubule drug
(100 /xM demecolcine, 50 fxM podophyllotoxin, 50 /xM
vincristine, 50 /xM tubulozole and 10 /xM taxol) which
produced about 80 % inhibition (IC80) , followed by
stimulation with 10 /xM nicotine in the presence of each drug. Maximum inhibition for all drugs (80 - 90 %
inhibition) occurred quickly, within 10 - 30 min (Fig. 6).
In fact, an inhibition of 55 - 80 % can be obtained with these drugs upon simultaneous addition of the microtubule drug with nicotine.
Reversal of the effects of the microtubule drugs on nicotine-stimulated catecholamine release
It was also important to determine whether the inhibitory effects of these microtubule agents on nAChR- modulated catecholamine release could be reversed. Cultured bovine adrenal chromaffin cells were preincubated for 60 min at 37°C with 100 /xM demecolcine, 50 ixM podophyllotoxin, 50
/xM vincristine, 50 fxM tubulozole or 10 fxM taxol. The cells were then incubated in drug-free buffer for various time periods (2, 10, 30, 60 or 120 min) with 3 buffer changes, 70
1 2 0 CO -< LU 1 0 0
c l C Lul o 80 X CL m CL 60 LU JC c -iH ■ DEM (100 MM) CL 40 • POD (50 mM) LU CL ▲ TUB (50 /(M) O 20 ♦ VCR (50 HM)
CO ...... J i L 0 0 10 20 30 40 50 60 180 PRETREATMENT TIME (min)
FIGURE 6. Time-dependent effects of microtubule drugs on nAChR-stimulated [H]norepinephrine release. Cells were pretreated for the indicated times with 100 /zM demecolcine (DEM) , 50 juM podophyllotoxin (POD) , 50 /zM tubulozole (TUB) or 50 /zM vincristine (VCR) prior to stimulation with 10 /zM nicotine in the continued presence of the drug. Results are expressed as a percentage of the inhibition of the control 10 /zM nicotine-stimulated response. In the absence of any microtubule drug, basal [3H]norepinephrine fractional release was 0.056 ± 0.003 and the fractional release caused by 10 /zM nicotine was 0.176 ± 0.008. Values represent means ± SEM of 6 experiments performed in triplicate. 71 followed by stimulation with 10 fiM nicotine for 10 min in
the absence of drug. Figure 7 illustrates the reversibility
of the inhibitory effects of these microtubule drugs on
nicotine-stimulated [3H]norepinephrine release. Within 2
min of removal of the microtubule drug, a reversal of the
inhibition of [3H]norepinephrine release is seen for all the
microtubule agents. With a 30 min washout period, all the
microtubule drugs had approximately a 2-fold greater
stimulation of [3H]norepinephrine release by nicotine when
compared to cells in which the drugs had not been washed
out. Over >80 % of nAChRs function returns when chromaffin
cells are drug-free for 60 min or longer.
Effect of chronic treatment with microtubule drugs on adrenal catecholamine release
Because of the relatively rapid onset and reversibility
of the inhibitory action of microtubule drugs on nAChR-
mediated catecholamine release, it was of interest to
determine if longer treatment times with lower
concentrations of microtubule drugs can still inhibit
secretion. Cells were exposed with approximately IC10
concentrations of the microtubule drugs for a 22 hr period,
loaded with [3H]norepinephrine and stimulated in the presence of the microtubule drug for a total preincubation time with the drug of 24 hr. Vincristine (0.1 (jlM) , podophyllotoxin (0.3 /iM) and demecolcine (0.8 fj,M) caused small, but significant, decreases of 28 %, 34 % and 30 % on 72
120 CO - c
1 0 0 cmLU 80 o c_ cm -M =c C Q_ o 60 o ■DEN (100 * ) 40 APOD (50 *N) •TAX (10 AN) cm OTUB (50 AH) o 20 □VCR (50 aH)
co Q i .... i . .» » 1 * » * » 1 * ■ * ■ 1 ■ * * ■ 1 » » ■ ■ 1 11 i 0 10 20 30 40 50 60 120 WASH PERIOD (min)
FIGURE 7. Reversal of the effects of microtubule drugs on nAChR-stimulated [3H]norepinephrine release from cultured bovine adrenal chromaffin cells. Cells were pretreated for 1 hr with 100 jiM demecolcine (DEM) , 50 /xM podophyllotoxin (POD) , 10 juM taxol, 50 /xM tubulozole (TUB) or 50 |XM vincristine (VCR), washed with PSS and incubated in PSS (in the absence of microtubule drug) for the indicated times prior to stimulation with 10 /xM nicotine. Results are expressed as a percentage of the control 10 /xM nicotine- stimulated response. In the absence of any microtubule drug, basal [3H]norepinephrine fractional release was 0.074 ± 0.005 and the fractional release caused by 10 /xM nicotine was 0.237 ± 0.013. Values represent means ± SEM of 5 to 6 experiments performed in triplicate. 73 nicotine-stimulated [3H]norepinephrine release, but did not
affect release stimulated by 56 mM KC1 (Fig. 8).
Vinblastine (0.1 /xM) and tubulozole (0.2 jxM) , also,
decreased [3H]norepinephrine release, but the effect was not
significant (Fig. 8). None of the microtubule drugs
affected basal catecholamine release under these chronic
treatment conditions (Table 4).
Effects of microtubule drugs on the microtubular network of adrenal chromaffin cells
To determine if these agents also alter adrenal
microtubules under similar conditions in which they inhibit
nAChR-mediated secretion of [3H]norepinephrine, the direct
effects of the microtubule drugs on the microtubular network of cultured adrenal chromaffin cells were examined using
immunocytochemical techniques (Fig. 9, A-F and Fig. 10, A-
F). When cultured adrenal chromaffin cells were stained with anti-tubulin antibodies and examined under a fluorescence microscope, control cells had an evenly dispersed microtubule network radiating throughout the cell bodies and down the length of the neurites (Fig. 9A). Cells treated for 60 min with the vinca alkaloid, vincristine (10
/xM), lack a discernable microtubular network but instead contained tubulin paracrystals in the cytosol (Fig. 9B).
These cells were also less fluorescent than control cells.
In cultured bovine adrenal chromaffin cells treated for one hr with taxol (10 /xM), highly fluorescent parallel arrays 74
Table 4. Effects of a 24 hr treatment with low concentrations of microtubule drugs on basal (nonstimulated) catecholamine release.3
Basal Catecholamine Release3
Basal (control) 0.076 + 0.009
+ Demecolcine (0.8 MM) 0.073 + 0.007
+ Podophyllotoxin (0.3 mM) 0.077 + 0.006
+ Tubulozole (0.2 MM) 0.097 + 0.022
+ Vinblastine (0.1 MM) 0.082 + 0. 006
+ Vincristine (0.1 MM) 0.094 + 0. 010 aCultured adrenal chromaffin cells were treated for 24 hr in the absence (control) or presence of the indicated microtubule drug. The amount of [3H]norepinephrine was determined during a 10 min period. Results are expressed as a fraction of the total incorporated [3H]norepinephrine released. Values represent means ± SEM of 5 to 7 experiments. None of the treatments were statistically different from control (p < 0.05). 75
|SECflETAGOGt£ S tub (0.20 0)
to .16 0VBL (0.10 0) □ p o o (0.30 0) 0VCR (0.12 0) □ D E M (0.80 * ) oc i .12
3 S S .08
.04
CO
NICOTINE (10 mM) K* (56 mM)
FIGURE 8. Effect of chronic treatment with microtubule drugs on adrenal nicotine- and K+-stimulated [3H]norepinephrine release. Cells were pretreated with 0.1 /xM vinblastine (VBL) , 0.1 nH vincristine (VCR), 0.2 /xM tubulozole (TUB), 0.3 /xM podophyllotoxin (POD) or 0.8 /xM demecolcine (DEM) for 24 hr prior to stimulation with either 10 /xM nicotine or 56 mM KC1 in the continued presence of the drug. Results are expressed as a percentage of the respective secretagogue-stimulated control response. Basal [3H]norepinephrine fractional release was 0.069 ± 0.001 and stimulated fractional releases were 0.232 ± 0.011 and 0.140 ± 0.008 for nicotine and KC1, respectively. Results significantly different from control are marked by an asterisk (p < 0.05). Values represent means ± SEM of 6 to 12 experiments performed in triplicate. FIGURE 9. Effects of various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-tubulin antibodies. A: Control cells. B to F: Cells treated for 60 min with 10 /iM vincristine (B), 10 /iM tubulozole (C) , 100 jiM podophyllotoxin (D) , 100 fiM demecolcine (E) and 10 /iM taxol (F). Calibration bar = 20 Aim. Results are representative of 4 to 7 experiments.
76 77
t
Figure 9 Figure 9 continued 79
Figure 9 continued 80
were seen scattered throughout the cytosol. The neurites of
these cells are difficult to discern due to reduced
fluorescence (Fig. 9F). One hr treatment with
podophyllotoxin (100 /xM) , tubulozole (10 juM) and demecolcine
(100 /xM) also results in disrupted microtubular networks
(Fig. 9, C-E). Cells treated with these drugs exhibit
decreases in total cell fluorescence, making it difficult to
detect neurites, and uneven distributions of fluorescence in
the cytoplasm. A more pronounced effect of the drugs on the microtubular network can be seen in cells treated with these
agents for 3 h (Figure 10, A-F).
Time-course for the appearance of vinblastine-induced paracrystals
Small alterations in the chromaffin cell microtubular network, produced by treatment with the microtubule drugs are difficult to detect due to the qualitative nature of tubulin immunofluorescence. However, to determine how early these drugs could alter microtubules, the time-dependent effects of vinblastine were investigated. Vinblastine was used in these studies since its effects are manifested by formation of easily detectable tubulin paracrystals. The time-course for the effects of vinblastine on adrenal microtubules is seen in Fig. 11, B-D. Cultured chromaffin cells were treated with 10 /xM vinblastine for 15, 60 and 180 min. In cells treated for 15 min, several tubulin paracrystals can be noticed scattered throughout the FIGURE 10. Effects of various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-tubulin antibodies. A: Control cells. B to F: Cells treated for 180 min with 10 /xM vincristine (B) , 10 /iM tubulozole (C) , 100 /xM podophyllotoxin (D) , 100 fiM demecolcine (E) and 10 /iM taxol (F). Calibration bar = 20 Hm. Results are representative of 4 to 7 experiments.
81 82
Figure 10 Figure 10 continued
c Figure 10 continued FIGURE 11. Time-course for the appearance of vinblastine- induced paracrystals in cultured adrenal chromaffin cells by anti-tubulin antibodies. A: Control cells. B to D: Cells treated with vinblastine for 15 min (B), 60 min (C) and 180 min (D) . Calibration bar = 10 [im. Results are representative of 4 to 8 experiments.
85 86
Figure 11 87
Figure 11 continued
k ■^v \ 88
cytoplasm and along the neurites. A progressive increase in
the number of tubulin paracrystals can be observed in the
cytosol as the treatment times increases. After 180 min of
treatment, the paracrystals are guite obvious and spread
throughout the adrenal cells. Therefore, with increasing
treatment time with vinblastine from 15 min, when only a few
paracrystals can be seen, up to a 3 hr exposure when many
are seen, there appears to be a time-dependent effect on the
disruption of the microtubular network.
Effect of microtubule drugs on the acetylated tubulin population of adrenal chromaffin cells
Various populations of microtubules have been
identified on the basis of posttranslational modifications.
One such population of microtubules are acetylated
microtubules. These microtubules have been shown to belong
to a stable group of microtubules, i.e., the rate of
depolymerization of these microtubules is slower than
unstable microtubules which are constantly polymerizing and depolymerizing. To determine if the microtubule drugs are altering a more stable population of microtubules, represented by acetylated microtubules, adrenal chromaffin cells were treated with microtubule drugs, stained with 6-
11B-1 (anti-acetylated tubulin antibodies) and examined by fluorescence microscopy. Control cells contained an evenly dispersed acetylated microtubular network, radiating from the perinuclear region and extending throughout the cell 89 bodies. However, the density of the microtubular network
was not as great as that seen with the tubulin antibodies.
In some cells the microtubular network extended down the
neurites (Fig. 12A). In cells treated for 60 min with 100
MM demecolcine, 100 jxM podophyllotoxin and 10 /xM tubulozole
(Fig. 12, B-D), the microtubules containing acetylated
tubulin had a wiry appearance rather than an evenly
dispersed microtubular network as seen in untreated cells.
The total fluorescence was less in microtubule drug-treated
cells than in control cells. Some cells that were treated
with 10 /xM taxol had highly fluorescent parallel wiry arrays
scattered in the perinuclear region of the cells (Fig. 12E) .
In other taxol-treated cells, less fluorescence was noted
and only a few microtubules were observed scattered
throughout the cytoplasm that had a wiry, curved appearance.
The neurites of these cells are difficult to discern due to
reduced fluorescence (Fig. 12E). Many cells treated with
the vinca alkaloid, vinblastine (10 /xM), had paracrystals present in the cytosol (Fig. 12F). However, other vinblastine-treated cells did not have paracrystals but,
like demecolcine- and podophyllotoxin-treated cells, the acetylated microtubules were curved and wiry in appearance.
The overall fluorescence in cells treated with vinblastine was less than control cells. Similar results were obtained for vincristine (10 /xM) treated cells (Fig. 12G). The acetylated microtubules present were wiry as in other FIGURE 12. Effects of various microtubule drugs on the immunological staining of cultured adrenal chromaffin cells by anti-acetylated tubulin antibodies (6-11B-1). A: Control cells. B to G: Cells treated for 60 min with 100 /xM demecolcine (B) , 100 /xM podophyllotoxin (C) , 10 mM tubulozole (D), 10 /xM taxol (E), 10 /xM vinblastine (F) and 10 /zM vincristine (G) . Calibration bar = 20 fim. Results are representative of 3 to 5 experiments.
90 91
Figure 12 92
Figure 12 continued
D Figure 12 continued Figure 12 continued 95
microtubule drug-treated cells.
Effect of microtubule drugs on [3H]nicotine binding to rat brain membrane fragments
Microtubule drugs inhibit only nAChR-mediated
catecholamine release, as demonstrated above. Therefore, it
is possible that these drugs can act either as cholinergic
antagonists or as microtubule disrupting agents. To
determine if the action of the microtubule drugs on nAChRs
is due to interference with agonist binding, the ability of
these compounds to interfere with [3H]nicotine binding to
nAChR binding sites was examined. Membrane fragments from
rat brain were used as a plentiful source of neuronal
nAChRs. Competition curves of specific [3H]nicotine binding
by the nAChR agonists, nicotine and carbachol, are depicted
in Fig. 13. Both nicotine (appl^ = 14 nM) and carbachol
(appK£ = 530 nM) inhibited [3H]nicotine binding,
demonstrating the presence of nAChRs in this preparation.
At concentrations of the microtubule drugs known to inhibit
70 - 80 % of nicotine mediated release (50 fiM concentrations
of each), demecolcine, podophyllotoxin, tubulozole,
vincristine and nocodazole did not block [3H]nicotine binding (Fig. 14). In addition, 50 juM adiphenine (a noncompetitive nAChR ion channel blocker), failed to block
[3H]nicotine binding. These results suggest that the microtubule drugs are not acting as nAChR antagonists in rat brain membrane fragments. It should be pointed out, though, 96
CD Z I—i CARBACHOL o z ^ NICOTINE 1—1 o GO • rH M— LU •rH O i—I CD f— Q_ o in CD l—l Z V -- ✓ \ 1 1 \ =n CO
1 1 ■■■■■■•! ■ 1 lllluJ ■ ■ » “ “ J • ......
10" 910“ 810" 710" 610" 510" 410"3 [DRUG] (M)
FIGURE 13. Drug displacement curves of specific [3H]nicotine binding by unlabelled nicotine and carbachol. Aliquots of membrane protein were incubated with [3H]nicotine (15 nM) in the presence and absence of various concentrations of unlabelled nicotine and carbachol for 60 min at room temperature. Carbachol (1 mM) was used to determine nonspecific binding which was 52 % of total binding. Control specific [3H]nicotine binding was 7200 ± 1800 CPM/mg protein (mean ± SEM). Results are expressed as a percentage of specific [3H]nicotine binding. Values represent the mean ± SEM of 3 to 4 experiments performed in duplicate. 97
120 I I o 100 ± z I I ZD I O / -- N GO o 80 -rH LlJ 4- z : •rH i—i o i— <1> 60 o C l o i/) 1—1 a-s 40 = c CO 20
0 CONTROL DEM POD TUB VCR NOC ADI
FIGURE 14. Effect of various microtubule drugs on [3H]nicotine binding in rat brain membrane fragments. Aliquots of membrane protein were incubated with [3H]nicotine (10 nM) for 60 min at room temperature in the presence of 50 /uM concentrations of demecolcine (DEM), podophyllotoxin (POD), tubulozole (TUB), vincristine (VCR), nocodazole (NOC) or adiphenine (ADI). Carbachol (1 mM) was used to determine nonspecific binding which was 52 % of total binding. Control specific binding was 53 00 ± 900 CPM/mg protein. Results are expressed as a percentage of the control specific [3H]nicotine binding. Values represent the mean ± SEM of 8 to 10 experiments performed in duplicate. 98 that this rat brain preparation lacks an intact
cytoskeleton.
Effect of microtubule drugs on [3H]nicotine binding to nAChRs in intact adrenal chromaffin cells
In membrane fragments which lack an intact microtubular
network, microtubule drugs had no effect on [3H]nicotine
binding. Therefore, to determine if an intact microtubular
network is necessary for the action of the microtubule
drugs, [3H]nicotine binding studies were conducted on intact
adrenal chromaffin cells. Additionally, the effects of
various cholinergic drugs (carbachol, hexamethonium,
mecamylamine, d-tubocurarine and adiphenine) on [3H]nicotine
binding were also examined. A 50 /xM concentration of
carbachol, hexamethonium, mecamylamine, d-tubocurarine and
adiphenine significantly inhibited [3H]nicotine binding by
85 %, 73 %, 66 %, 53 % and 64 %, respectively (Fig. 15) .
The effects of various microtubule drugs, at concentrations
shown to inhibit 90 % of nicotine-stimulated catecholamine
release, on [3H]nicotine binding in intact cultured bovine
adrenal chromaffin cells were also examined. At 50 ixK
concentrations, demecolcine, podophyllotoxin, taxol,
vinblastine and vincristine significantly inhibited
[3H]nicotine binding by 55 %, 50 %, 48 %, 62 % and 39 %,
respectively (Fig. 15). Since it has been shown that cold temperature (0°C) causes disruption of microtubules
(Penningroth and Kirschner, 1977), binding was also 99
160 140 o z I ID 120 o CD o •rH 100 LxJ 4- Z •i—l l— l o t — . I 20 I
0 CONTROL CCH C6 MMA d-TC ADI OEM POO TAX TUB VBL VCR 0°C
FIGURE 15. The effect of various microtubule drugs on [3H]nicotine binding to intact primary cultures of adrenal chromaffin cells. Cell cultures (lxlO6 cells/well) were incubated with 10 nM [3H]nicotine for 70 min at 37°C in the presence or absence of demecolcine (DEM), podophyllotoxin (POD), taxol (TAX), tubulozole (TUB), vinblastine (VBL), vincristine (VCR), carbachol (CCH), hexamethonium (C6), mecamylamine (MMA), d-tubocurarine (d-TC) or adiphenine (ADI). Unlabeled nicotine (50 /iM or 1 mM) was used to determine nonspecific binding which was 56 % of total binding. The mean value for control binding was 9600 ± 2300 CPM/mg protein. Results significantly different from specific binding in the absence of drug, are marked by asterisks (** p < 0.01, * p < 0.05). Values represent the mean ± SEM of 3-12 experiments performed in triplicate. 100
conducted at 0°C to examine the effects on [3H]nicotine
binding. The results obtained suggest that cold temperature
has no effect on binding, however, if indeed alterations in
the microtubular network interferes with [3H]nicotine
binding, as observed with the microtubule drugs, then it is
possible that the conditions used in these studies were not
adequate enough to disrupt microtubules or to detect an
effect on binding (Fig. 15) .
Noncompetitive inhibition of nicotine-stimulated catecholamine release by microtubule drugs
In bovine adrenal chromaffin cells, taxol, vinblastine and colchicine have been shown to exhibit properties similar to those of noncompetitive inhibitors of the nAChR- associated ion channel (McKay et a l ., 1985). Therefore, to determine the type of inhibitory action that demecolcine, podophyllotoxin, tubulozole and vincristine have on adrenal nAChRs, the concentration-response relationship of nicotine on catecholamine secretion was determined in the presence and absence of the microtubule drugs. In the absence of microtubule drugs, maximum stimulation of catecholamine release occurred with approximately 50 /xM nicotine and half maximum stimulation occurred with 5 /xM nicotine (Fig. 16,
Table 5). In the presence of a fixed concentration of demecolcine (20 /xM) , podophyllotoxin (5 /xM) , tubulozole (5
/xM) and vincristine (5 juM) , no changes in the concentration of nicotine eliciting half-maximum response (EC50 values) 101
Table 5. Effects of microtubule drugs on the concentration- response effects of nicotine on adrenal catecholamine release3.
EC50 Er,axb
Nicotine 5.0 + 0.5 0.176 + 0.018
+ Demecolcine (20 /xM) 4.4 ± 0.4 0.116 + 0.015
+ Podophyllotoxin (5 /xM) 4.3 + 0.3 0.115 + 0.014
+ Tubulozole (5 /iM) 6.2 + 1.0 0.109 + 0. 016
+ Vincristine (5 juM) 7.0 + 3.3 0.109 + 0. 016' aHalf maximum effective concentrations (EC50) of nicotine (/xM) were calculated as described in Material and Methods from the data found in Fig. 16. Values represent means ± SEM (n=5-7). bEmax is the maximum response obtained for nicotine response in the presence or absence of the microtubule drug. cSignificantly different from the corresponding secretagogue-stimulated controls (p < 0.05). 102
CO ONIC • NIC + OEM (20 jM) LU O' ANIC ♦ POO (5 *) LU A NIC + TUB (5 aM) o □ NIC ♦ VCR ( 5 ») o : c_ n : c CL o LU o
Od O =c CO 10"6 10-5 10"4 [NICOTINE] (M)
FIGURE 16. The concentration-response effects of nicotine on adrenal catecholamine release in the absence and presence of various microtubule drugs. Cultured adrenal chromaffin cells were pretreated for 30 min with either 20 /zM demecolcine (DEM) , 5 juM podophyllotoxin (POD) , 5 /zM tubulozole or 5 /zM vincristine (VCR) prior to stimulation with increasing concentrations of nicotine in the continued presence of the drug. Control cells were treated identically except in the absence of any microtubule drug. Results are expressed as a percent of 10 juM nicotine- stimulated [3H]norepinephrine released during a 10 min stimulation period. In the absence of any microtubule drug, basal [3H]norepinephrine fractional release was 0.037 ± 0.002 and the fractional release caused by 10 /zM nicotine was 0.2 00 ± 0.004. Values represent means ± SEM of 6 to 7 experiments performed in triplicate. 103 were observed (Table 5). However, the maximum amount of
[3H]norepinephrine secretion (Emax) was reduced by 53 %, 59
%, 45 % and 43 % by demecolcine, podophyllotoxin, tubulozole
and vincristine, respectively (Table 5). The type of
inhibition of the microtubule drugs appears to be
noncompetitive.
Effect of Triton-extraction on [125I]mAb 35 binding to adrenal chromaffin cells
Evidence exists for the association of muscle nAChRs with the cytoskeleton (Prives et a l ., 1982; Stya and
Axelrod, 1983; Phillips et al., 1991; Froehner et al.,
1991). Results acquired from earlier studies and work done in our laboratory suggests that adrenal nAChRs might also be associated with the Triton-insoluble cytoskeleton.
mAb 35 is a monoclonal antibody raised against nAChRs from Electrophorus electric organ, that recognizes the main immunogenic region (MIR) of muscle and electric organ AChRs
(Tzartos, 1981) and cross reacts with chick ciliary ganglion nAChRs (Jacob et a l ., 1984) and the nAChRs from adrenal chromaffin cells (Higgins and Berg, 1987). Hybridoma cells, which secrete monoclonal antibodies (mAb 35) reactive with nAChRs, were grown and maintained in culture as described by
Harlow and Lane (1988) until a desired amount of supernatant containing mAb 35 was obtained. mAb 35 was purified by ammonium sulfate precipitation from hybridoma culture media
(Harlow and Lane, 1988). To provide evidence that the 104
purified antibody was selective for adrenal chromaffin
nAChRs, the ability of mAb 3 5 to modulate the secretory
response of chromaffin cells to nicotine and depolarizing
concentrations of K+ were examined using concentrations of
mAb 35 reported by Higgins and Berg (1987) to attenuate
nicotine-stimulated release by 40 % and 90 %. Exposure of
chromaffin cells to 10 nM and 100 nM of mAb 35 for 24 hr
significantly inhibited nicotine-stimulated release by 70 %
and 90 %, respectively. However, these same concentrations
of mAb 35 did not affect catecholamine release by 56 mM KC1
(Fig. 17). These results verify that the monoclonal
antibody purified (mAb 35) is selective for nAChRs.
In these studies mAb 3 5 was radioiodinated and used as a probe to detect adrenal nAChRs associated with the Triton- insoluble cytoskeleton. Triton is a mild detergent that removes lipids and soluble proteins and leaves the internal cytoskeleton and associated proteins essentially intact
(Ben-Ze'ev et al., 1979). Therefore, to determine if adrenal nAChRs are indeed associated with the cytoskeleton,
[125I]mAb 3 5 was used to label nAChRs. The degree of association of nAChRs with the cytoskeleton was determined as described in the Material and Methods section. In cells which were not Triton-extracted, 19 fmole of [125I]mAb 3 5 labeled nAChRs were associated with surface sites (Fig. 18,
Table 6). After Triton-extraction 7 fmoles of nAChRs remained associated with the Triton-insoluble cytoskeleton. 105
LU CO NICOTINE (10 *M) K+ (56 mM) FIGURE 17. Effect of 24 hr exposure with mAb 35 on secretagogue-stimulated adrenal [3H]norepinephrine release. Cells were exposed to either 10 nM or 100 nM mAb 3 5 for 24 hr prior to stimulation with either 10 /xM nicotine or 56 mM KC1 in the absence of mAb 35. Results are expressed as a fraction of the total incorporated [3H]norepinephrine released during the 10 min stimulation period. Basal [3H]norepinephrine fractional release was 0.075 ± 0.005, whereas stimulated fractional releases were 0.212 ± 0.006 and 0.078 ± 0.012 for 10 juM nicotine and 56 mM KC1, respectively. Values represent means ± SEM of 2 experiments performed in triplicate. Results significantly different from the corresponding secretagogue-stimulated control are marked by an asterisk (p < 0.05). 106 Table 6. [125I]mAb 35 binding to Triton-extracted cultured adrenal chromaffin cells.® [125I]mAb 35 Bound Receptors % (fmole/106 cells) per cell Control Control Cells 19 5909 100 (unextracted) Triton-Insoluble 7b 2177b 36b Cell Extract aCultured adrenal chromaffin cells (lxlO6 cells/well) were incubated with [125I]mAb 35 (5 nM) for 60 min at 37°C. After washing unbound [125I]mAb 35, the cells were extracted for 5 min with buffer containing 0.5 % Triton. Unlabeled mAb 35 (0.5 /iM) was used to determine nonspecific binding which was 20 % of total binding. The mean value for control was 652 ± 40 CPM/lxlO5 cells. Values represent the mean ± SEM of 3 experiments performed in triplicate and were analyzed by Two Way ANOVA. The amount of protein per well was 0.2 2 ± 0.01 mg. bSignificantly different from control (p < 0.01). 107 o in o QQ in Q_ c\i u SURFACE BINDING TRITON-INSOLUBLE BINDING FIGURE 18. [125I]mAb 35 binding to Triton-extracted cultured adrenal chromaffin cells. Cultured cells (lxlO6 cells/well) were incubated with [125IlmAb 35 (5 nM) for 60 min at 37°C. After washing unbound [l25I]mAb 35, the cells were extracted for 5 min with buffer containing 0.5 % Triton. Unlabelled mAb 35 (0.5 (jlM) was used to determine nonspecific binding which was 20 % of total binding. The mean value for control was 652 ± 40 CPM/lxlO6 cells. Values represent the mean ± SEM of 3 experiments performed in triplicate. Results significantly different from control (total specific binding in the absence of drug) are marked by an asterisk (** p < 0.01, Two Way ANOVA). The amount of protein per well was 0.22 ± 0.01 mg. 108 These results suggest that 36 % of the total surface receptors are associated with the Triton-insoluble cytoskeleton. Influence of microtubule drugs on the association of adrenal nAChRs to Triton-extracted adrenal chromaffin cells To determine if an alteration in microtubules alters nAChRs association with the cytoskeleton, the effect of microtubule drugs on the Triton-extractability of nAChRs were investigated. Additionally, the effects of various cholinergic ligands (nicotine, mecamylamine and adiphenine) on [125I]mAb 35 binding were also examined. At concentrations of nicotine (10 /xM) , mecamylamine (1 yuM) and adiphenine (5 /xM) shown to inhibit 80 % of nicotine- stimulated catecholamine release in chromaffin cells, only nicotine and mecamylamine significantly decreased [125I]mAb 35 binding (24 % and 51 %, respectively), Fig. 19, Table 7. The noncompetitive nAChR ion channel blocker, adiphenine, had no significant effect on the amount of [125I]mAb 35 (7 fmole) associated with the Triton-insoluble cytoskeletal component (Fig. 19, Table 7). At concentrations of the microtubule drugs known to inhibit 70 - 80 % of nicotine- stimulated secretion, demecolcine podophyllotoxin, vinblastine, vincristine and taxol significantly decreased the Triton-insoluble bound [125I]mAb 35 by 37 %, 46 %, 33 %, 38 % and 43 %, respectively (Fig. 19, Table 7). Although tubulozole inhibited [125I]mAb 35 binding by 24 % it was not Table 7. Effect of microtubule drugs on [125I]mAb 35 bound to Triton-extracted adrenal chromaffin cells.3 Receptor [125I]mAb 35 Bound per cell (fmole/lxlO5 cells) (x 103) % Control Unextracted Cells 17 + 0.70 5.6 ± 0.1 Control, Triton-Insoluble 7 + 0.30 2.3 ± 0. 08 100 Cell Extract + Nicotine (10 /iM) 5 + 0. 3b 1.7 ± 0. 09b 76 ± 6b + Mecamylamine (1 /iM) 4 + 0. 4b 1.1 ± 0. 10b 49 ± 5b + Adiphenine (5 /xm) 7 ± 0. 6b 2.2 ± 0.20b 95 ± 6b + Demecolcine (50 pM) 5 ± 0. 5b 1.4 ± 0.16b 63 ± 5b + Podophyllotoxin (50 pM) 4 ± 0. 7b 1.2 ± 0.20b 54 ± 7b + Taxol (30 /jiM) 5 ± 0. lb 1.5 ± 0.02b 67 ± 2b + Tubulozole (50 pM) 6 ± 1. 5b 1.7 ± 0.50b 76 ± 20b + Vinblastine (50 /xM) 5 ± 0. 6b 1.4 ± 0.20b 62 ± 8b + Vincristine (50 jiM) 4 ± 0. 3b 1.3 ± 0.10b 57 ± 3b aCultured adrenal chromaffin cells (lxlO6 cells/well) were incubated with [125I]mAb 35 (5 nM) for 60 min at 37°C in the presence or absence of microtubule drug, receptor agonist, antagonist or receptor ion channnel blocker. After washing unbound [ 25I]mAb 35, the cells were extracted for 5 min with buffer containing 0.5 % Triton. Unlabeled mAb 35 (0.5 nM) was used to determine nonspecific binding, which was 20 % of total binding. The mean value for control was 249 ± 9 CPM/lxlO° cells. Values represent the mean ± SEM of 3 experiments performed in triplicate and were analyzed by Two Way ANOVA. The amount of protein per well was 0.22 ± 0.01 mg. bSignificantly different from control (p < 0.01). 110 300 r U) o CD 0 m o co vO o «< I E in 2 8- CONTROL NIC NEC ADI DEM POO TAX TUB VBL VCR FIGURE 19. Effect of microtubule drugs on [125I]mAb 35 binding to Triton-extracted adrenal chromaffin cells. Cultured adrenal chromaffin cells (lxlO6 cells/well) were incubated with 50 /iM [125I]mAb 35 for 60 min at 37°C in the presence the presence or absence of demecolcine (DEM), podophyllotoxin (POD), taxol (TAX), tubulozole (TUB), vinblastine (VBL), vincristine (VCR), carbachol (CCH), hexamethonium (C6), mecamylamine (MMA), d-tubocurarine (d- TC) or adiphenine (ADI). After washing unbound [125I]mAb 35, the cells were extracted for 5 min with buffer containing 0.5 % Triton. Nonspecific binding was 20 % of total binding. The mean value for control Triton-insoluble bound [125I]mAb 35 was 249 ± 9 CPM/lxlO6 cells. Values represent the mean ± SEM of 3 experiments performed in triplicate. Results significantly different from control are marked by asterisks (** p < 0.01). The amount of protein per well was 0.22 ± 0.01 mg. I l l statistically (Two Way ANOVA) significant (Fig. 19). Effect of microtubule drugs on [125I]mAb 35 binding to intact adrenal chromaffin cells In the previous study, cholinergic ligands (nicotine and mecamylamine) and microtubule drugs decreased the association of [125I]mAb 35 with the Triton-insoluble N cytoskeleton. Therefore, to determine if the reduction in binding was due to an inhibitory effect of these compounds on [125I]mAb 35 binding, binding studies were conducted on intact adrenal chromaffin cells. In these studies, none of the drugs (10 /xM nicotine, 1 /xM mecamylamine, 50 /xM demecolcine, 50 /xM podophyllotoxin, 50 jxM tubulozole, 30 /xM taxol, 50 juM vinblastine or 50 jxM vincristine) affected [125I]mAb 35 binding to intact (non-extracted) chromaffin cells (Table 8). Table 8. Effect of microtubule drugs on [125I]mAb 35 binding to cultured adrenal chromaffin cellsa Receptor [125I]mAb 35 Bound per cell (fmole/lxlO6 cells) (x 103) % Control Control (unextracted) 15 + 2 4.7 + 0.6 100 + Nicotine (10 jxM) 16 + 2 4.8 + 0.5 107 ± 18 + Mecamylamine (1 /xM) 15 + 1 4.5 + 0.1 99 ± 14 + Demecolcine (50 /xM) 19 + 4 5.8 ± 1.1 126 ± 27 + Podophyllotoxin (50 /xM) 18 + 3 5.7 + 1.0 121 ± 18 + Taxol (30 /xM) 17 + 1 5.2 + 0.5 112 ± 8 + Tubulozole (50 jiM) 15 + 2 4.8 + 0.5 102 ± 14 + Vinblastine (50 /xM) 17 + 3 5.3 + 1.0 111 ± 16 + Vincristine (50 /xM) 17 + 2 5.3 + 0.7 114 ± 15 aCultured adrenal chromaffin cells (lxio6 cells/well) were incubated with [125I]mAb 35 (5 nM) for 60 min at 37°C in the presence or absence of microtubule drugs, receptor agonist or antagonist. Unlabeled mAb 35 (0.5 jxM) was used to determine nonspecific binding, which was 20 % of total binding. The mean value for control was 652 ± 40 CPM/lxlO6 cells. Values represent the mean ± SEM of 3 experiments performed in triplicate and were analyzed by Two Way ANOVA. None of the treatments were statistically different from control (p< 0.05). The amount of protein per well was 0.22 ± 0.01 mg. CHAPTER V DISCUSSION Chromaffin cells contain a cytoskeletal network made up microtubules and other filaments. In various cells these proteins have been implicated in playing a role in cellular secretion. This is based on the ability of microtubule drugs, such as colchicine and vinblastine, to inhibit specific exocytotic processes. In adrenal chromaffin cells, evidence exists that nAChRs are associated with cytoskeletal proteins and that this association may be important for receptor function (McKay and Schneider, 1984; McKay, 1988). This is based on the effects of taxol and vinblastine on catecholamine release from cultured adrenal chromaffin cells. The precise mechanism by which these drugs inhibit receptor function is not established. Therefore, the major objectives of these studies were to determine 1) the site and mechanism of action of the microtubule drugs, 2) if adrenal nAChRs are associated with microtubules and 3) if this association plays a role in receptor function. It is well documented that the microtubule drugs taxol, colchicine and vinblastine inhibit adrenal catecholamine release (Poisner and Bernstein, 1971; Douglas and Sorimachi, 1972; Trifaro, et a l ., 1972; McKay and Schneider, 1984; 113 McKay et al., 1987). Several steps in the secretory process are potential targets for these drugs. Classically, drugs which interfere with microtubular function have been shown to inhibit secretion by interfering with the release of secretory granules. This inhibitory effect has been demonstrated for the release of insulin from 6-cells of the pancrease (Lacey et a l ., 1968) and histamine release from mast cells (Gillespie et a l ., 1968) and basophils (Levy and Carlton, 1969). In adrenal chromaffin cells, vinblastine and taxol inhibit adrenal catecholamine release by mechanisms which do not involve the more terminal steps in stimulus-secretion coupling, secretory granule transport or exocytosis (McKay and Schneider, 1984). The site of action of taxol and vinblastine appears to be at the level of the adrenal nAChR. However, the mechanism by which these drugs inhibit nAChR function has not been determined. Reports in the literature suggest that vinblastine possesses anti-nAChR activity in neuronal tissues (Trifaro, 1972; McKay and Schneider, 1984; McKay et a l ., 1985). Several studies support the possibility that taxol, colchicine and vinblastine possess anticholinergic activity (Trifaro et al., 1972; McKay and Schneider, 1984; McKay et a l ., 1987). Other studies suggest that the microtubule drugs affect receptor function by an indirect action on microtubules (McKay and Schneider, 1984; McKay, et a l ., 1985; McKay, 1988) . 115 In the present study, the action of other microtubule drugs on nicotine-stimulated catecholamine release was investigated. The results of these studies demonstrate that all of the microtubule drugs (demecolcine, podophyllotoxin, tubulozole and vincristine) inhibit release. The effect of the microtubule drugs on secretion stimulated by other secretagogues was then examined. All of the microtubule drugs selectively inhibited nicotine stimulated catecholamine release. The microtubule drugs had little or no effects on secretion stimulated by 1) the nonreceptor- mediated secretagogues, K+, Ba+2, or veratridine or 2) by the receptor-mediated secretagogue, histamine. None of the drugs affected basal release of catecholamines. McKay and Schneider (1984) have reported similar effects for taxol and vinblastine on adrenal catecholamine release. Douglas et a l . (1965) have demonstrated that a common secretory pathway exists in chromaffin cells for different secretagogues, including ACh and depolarizing concentrations of K+. Therefore, if, indeed, a common secretory pathway exists, then the site of action of the microtubule drugs must not be at the terminal steps of exocytosis since microtubule drugs do not attenuate secretion stimulated by non-nAChR mechanisms. In these studies, the demonstration that none of the microtubule drugs affected Ba+2-stimulated release suggests that they do not directly alter secretory steps which might 116 involve calcium. Ba+2 stimulates adrenal catecholamine release by entering the cells through voltage operated calcium channels (VOC) and displacing calcium from intracellular storage sites (Hamano et al., 1991). In addition, the fact that the microtubule drugs did not alter release by depolarizing concentrations of K+, a step which preceeds opening of the VOC, suggests that the site of action of these drugs is at a step prior to cell depolarization. The lack of an inhibitory effect of the microtubule drugs on veratridine-stimulated catecholamine secretion also implies that the drugs are not directly affecting tetrodotoxin-sensitive Na+ channels, but supports an action of the drugs at a step prior to depolarization. These studies demonstrate that the microtubule drugs are inhibiting nAChR-stimulated release by interfering with signal transduction at the receptor level. Several lines of evidence support the hypothesis that the inhibitory effects of the microtubule drugs on nAChR function involves an action on microtubules. Firstly, all the microtubule drugs have similar effects, yet they are all structurally different and they interfer with microtubule function by different mechanisms. In addition, the IC50 values at which podophyllotoxin, tubulozole and vincristine inhibit adrenal catecholamine release are close to the IC50 concentrations which have been reported for each drug to inhibit in vitro tubulin polymerization, 0.5 - 1 tiM (Hoebeke and Van Nijen, 117 1975; Jordan and Farsell, 1983), 0.3 - 4 nM (Van Ginckel et al., 1984; Duanmy et al., 1989) and 0.1 - 2 nM (Owellen et a l . , 1972; Himes et a l ., 1977), respectively. It is interesting to note that the IC50 values for all the microtubule drugs are very close to values reported by McKay and Schneider (1984) for the inhibition of ACh-stimulated catecholamine release by taxol and vinblastine. These results would suggest, then, that the effect of these (structurally different) mictotubule drugs on catecholamine release is due to an action on microtubules, since they all have similar abilities to interact with microtubules. The rapid inhibitory effect on nAChR function observed in these studies, as well as the rapid reversal of drug action could also be explained by an action of the drugs on microtubules at or near the plasma membrane. It is well documented that the action of microtubule drugs on tubulin is quick and reversible (Manfredi and Horwitz, 1984; Hamel, 1990). In vitro tubulin binding constants reported for demecolcine, podophyllotoxin and vincristine are 7 - 14 juM (Banerjee and Bhattacharyya, 1979; Ray et a l ., 1981), 0.3 - 0.7 /iM (Flavin and Slaughter, 1974; Wilson et a l ., 1978) and 2 - 17 /iM (Owellen et a l ., 1972; Wilson, 1975), respectively. In the present study, maximum inhibitory effects produced by the microtubule drugs were observed within 10 min for demecolcine, vincristine, podophyllotoxin and tubulozole. Furthermore, the inhibitory effects of the 118 microtubule drugs were quickly reversible. Within 2 min after removal of the drug, a reversal of the inhibition of catecholamine release was observed. McKay and Schneider (1984) have also reported maximum inhibitory effects for taxol and vinblastine on nAChR-mediated catecholamine release to occur as early as 5 min. These results are similar to those reported for vincristine (< 5 min, Wilson, 1975), demecolcine (20 - 30 min, Banerjee et a l ., 1979; Ray et al., 1981), podophyllotoxin (< 30 min, Kelleher, 1977) and tubulozole (< 15 - 60 min, Geuens et a l ., 1985) for dissociation from tubulin. Adrenal chromaffin cells, like most cells, contain an extensive microtubular network (Bader et a l ., 1984). In these studies, all of the microtubule drugs have a direct effect on adrenal chromaffin cell microtubular network, and these effects are similar to those reported in other cells (Melmed et a l ., 1981; Manfredi and Horwitz, 1984; Mraz, 1985; Fujiwara and Tilney, 1975). If the receptor-related actions of the microtubule drugs involves microtubules, then it is important to establish that these drugs directly alter adrenal microtubules under similar conditions that affect receptor function. In the present study a 60 min treatment with concentrations of microtubule drug that inhibit receptor function was sufficient to detect the effects of the drugs on the adrenal microtubular system. Longer treatment times with the drugs demonstrated a greater effect 119 on the microtubular network. Treatments for shorter time periods were not conducted because of the qualitative nature of tubulin immunofluorescence technique; it is very difficult to detect small changes in the microtubular network. Since the functional effects of the microtubule drugs occur rapidly it was of interest to determine how early microtubule drugs alter microtubules. Therefore, the time-dependent effects of vinblastine were investigated. Vinblastine was used in these studies since its effects are manifested by formation of easily detectable paracrystals. Vinblastine-induced paracrystals could be detected as early as 15 min. An effect of microtubule drugs on the microtubular network as early as 15 min would agree well the time period the effects of the microtubule drugs on secretion occur. These results demonstrated that the microtubule drugs affect the adrenal microtubular network using similar drug treatment conditions which affect nAChR function, further supporting an action of the drugs on microtubules. Several different populations of microtubules have been identified within neurons and PC12 cells (Gozes and Sweadner, 1981; Black and Keyser, 1987). The heterogeneity of microtubules is due to posttranslational modifications. One such population of microtubules are acetylated microtubules. Acetylated microtubules comprise 10 - 40 % of the total cytoplasmic microtubule population (Maruta et al . , 120 1986). Various studies have shown that acetylation of microtubules increases the stability of microtubules, i.e., microtubules are more resistant to conditions which cause microtubule depolymerization (I^Hernault and Rosenbaum, 1983; Piperno and Fuller, 1985). The availability of a monoclonal antibody, 6-11B-1, which specifically recognizes the acetylated form of tubulin, has made it possible to study the distribution of microtubules containing acetylated tubulin and the effect of drugs on this population of microtubules (Piperno and Fuller, 1985). Immunofluorescence studies by Jasmin et al. (1990), using cryostat sections of chick anterior latissimus dorsi muscle, demonstrated that areas underneath motor plates which were labelled with FITC- conjugated a-Bgt were similar to areas labeled by the 6-11- B-l antibody. In these studies it was suggested that one possible role for microtubules localized in subsynaptic areas was in the transport of nAChRs to the endplated surface. To determine if adrenal chromaffin cells contain acetylated tubulin and if microtubule drugs alter this population of microtubules, cells were treated with the microtubule drugs, stained with 6-11B-1 (anti-acetylated tubulin antibodies) and examined by immunofluorescence microscopy. The results obtained from this study demonstrated that all of the microtubule drugs altered the acetylated microtubular network and suggest that the effect 121 of these drugs on nAChRs could be due to an alteration in the acetylated microtubule population. Another possibility for the effect of microtubule drugs on nAChR function is an anticholinergic action. Various reports in the literature demonstrate that taxol and vinblastine possess anti-nAChR activity (Trifaro et al., 1972; McKay and Schneider, 1984; McKay et al., 1985, McKay, 1988). In these studies the selectivity of the microtubule drugs for cholinergically mediated catecholamine secretion by isolated chromaffin cells, suggests that the inhibitory effect of the microtubule drugs on nAChR function might be due to a cholinergic action of the drugs. One approach to investigate this possibility is to determine whether the microtubule drugs interfere with binding of nicotine to nAChRs. To investigate the interaction of microtubule drugs with nAChRs, [3H]nicotine binding to membrane fragments from bovine adrenal medulla and cultured chromaffin cells was attempted. However, under the conditions used in these binding studies, specific binding could not be obtained from adrenal tissue or cell homogenates. There are various factors in the preparation of the membrane fragments that could render the nAChRs labile. A decrease in the number of nAChRs density could impede measurement of [3H]nicotine binding. It should also be pointed out that [3H]nicotine binding to membranes prepared from adrenal medullary tissue has not been reported in the literature. Binding of 122 [3H]nicotine to membrane fragments from cultured adrenal chromaffin cells was also attempted. Higgins and Berg (1988) have been the only group to report binding to membrane fragments from cultured adrenal chromaffin cells. However, attempts to duplicate their results have failed. Higgins and Berg (1988) investigated binding of various nAChR agonists and antagonists to membrane fragments from cultured bovine chromaffin cells and demonstrated that the affinity of these compounds were similar to those reported for [3H]nicotine binding to rat brain membrane fragments, suggesting a pharmacological similiarity between adrenal and brain neuronal nAChRs. Since [3H]nicotine binding to adrenal chromaffin membrane fragments could not be accomplished in our laboratory, membrane fragments from rat brain were used as a plentiful source of neuronal nAChRs. In rat brain homogenates, both carbachol and nicotine inhibited [3H]nicotine binding with appKi values close to those reported for high affinity nicotine binding to rat brain membrane (Romano and Goldstein, 1980; Lippiello and Fernandez, 1986) and chromaffin cell membranes fragments (Higgins and Berg, 1988), confirming the presence of nAChRs. The effects of microtubule drugs on [3H]nicotine binding were then examined. At concentrations of microtubule drugs known to inhibit approximately 80 % of nicotine-stimulated catecholamine release, none of the microtubule drugs 123 (demecolcine, podophyllotoxin, tubulozole, vincristine or nocodazole) inhibited [3H]nicotine binding. These results suggest that in rat brain membrane fragments, the microtubule drugs do not affect [3H]nicotine binding and therefore, do not appear to be nAChR antagonists. It should be pointed out that these rat brain preparations lack an N intact microtubule network. Similarly, McKay and coworkers (1985) have reported that the microtubule drugs taxol, vinblastine and colchicine do not inhibit [125I]a-Bgt binding to nAChRs from Torpedo electric organ. These results suggest that microtubule drugs are not classic nAChR antagonists and in order to see an action of the microtubule drugs on nAChRs an intact cytoskeletal network may be necessary. [3H]Nicotine binding studies were also conducted on cultured adrenal chromaffin cells. In this preparation, the cells are viable and nAChRs would remain coupled to the microtubular network. The microtubule drugs, demecolcine, podophyllotoxin, taxol, vinblastine and vincristine inhibited [3H]nicotine binding. Although tubulozole also blocked [3H]nicotine binding, the effect was not significant. These results suggest that in order for the microtubule drugs to affect [3H]nicotine binding an intact cytoskeleton is necessary. To directly determine if adrenal nAChRs are linked to the cytoskeleton, adrenal nAChRs were labelled using a 124 radiolabeled antibody, [125I]mAb 35. mAb 35, is a monoclonal antibody (mAb), raised against nAChR from Electrophorus electric organ that recognizes the main immunogenic region (MIR) of muscle and electric organ AChRs (Tzartos et al., 1981) and cross reacts with chick ciliary ganglion AChRs (Jacob et al., 1984) and nAChRs of bovine adrenal chromaffin cells (Higgins and Berg, 1987). After establishing that mAb 35 interacts specifically with adrenal nAChRs, chromaffin cells were incubated with 5nM [125I]mAb 3 5 for 1 hr prior to Triton-extraction. Under the conditions used in these studies, Higgins and Berg (1988b) have demonstrated that receptor internalization is minimum. The results obtained in the present study indicated that 3 6 % of surface nAChRs remain associated with the Triton- insoluble cytoskeleton. Similar findings have been reported for muscle nAChRs (Stya and Axelrod, 1983a). After establishing that a population of nAChRs remains associated with the Triton-insoluble cytoskeleton, the effects of various microtubule drugs (demecolcine, podophyllotoxin, taxol, tubulozole, vinblastine and vincristine) on the extractibility of the nAChRs were investigated. Triton is a mild detergent that removes lipids and soluble proteins and leaves the internal cytoskeleton and associated proteins essentially intact (Ben-Ze'ev et al., 1979). In these studies all of the microtubule drugs decreased the number of receptors 125 associated with the cytoskeleton as measured by the amount of [ 125I]mAb 35 bound. It is of interest to note that all the microtubule drugs decreased nAChR association with the Triton-insoluble cytoskeleton and [3H]nicotine binding to intact adrenal chromaffin cells to a similar extent, approximately 50 %. Since both nicotine and mAb 35 bind to different sites of the nAChRs, these results suggest that the microtobule drugs produce an alteration of the microtubular network that results in a major conformational change in the nAChR. Adiphenine, the noncompetitive blocker, had no effect on the extractability of nAChRs. A decrease in the number of receptors associated with the cytoskeleton was observed in cells treated with nicotine and mecamylamine. It is not known why this occured; however, a possible explanation may be that an interaction of drugs with the binding site causes a conformational change in the nAChR, altering receptor- cytoskeleton association and fascilitating the extractability of nAChRs. CHAPTER VI SUMMARY AMD CONCLUSIONS Cytoskeletal links with cell surface receptors occurs in a number of receptor systems and these linkages are involved with receptor topography and mobility and have been implicated in signal transduction mechanisms (Nicolson, 1976; Carraway and Carraway, 1989). Receptor-cytoskeletal associations have also been demonstrated for muscle nAChRs (Prives, et al., 1982; Stya and Axelrod, 1983a, 1983b; Connolly and Oldefin, 1985). In adrenal chromaffin cells, evidence exists that nAChRs are associated with cytoskeletal proteins and that this association may be important for receptor function (McKay and Schneider, 1984; McKay, 1988). In the present studies, the effects of several microtubule drugs on cultured adrenal chromaffin cells were investigated. The drugs used in these studies are structurally different but have one common feature: each drug interferes with microtubule function. All the microtubule drugs 1) selectively inhibited nAChR function, 2) inhibited nAChR function at concentrations reported for inhibition of tubulin polymerization and 3) altered microtubule networks under similar conditions at which they 126 127 inhibit nAChR function. Taken together, these studies suggested that the inhibitory effects of the microtubule drugs on nAChR-stimulated catecholamine release may be due to an action of the drugs on microtubules. These results support the hypothesis that adrenal nAChRs are functionally linked to the microtubular network. To directly explore the possibility that adrenal nAChRs are associated with the intracellular cytoskeletal network, detergent extraction experiments were conducted. The results of these studies demonstrated 1) that a population of adrenal nAChRs are associated with the Triton-insoluble cytoskeleton and 2) that microtubule drugs can alter the association of nAChRs with the cytoskeleton. Most importantly, the treatment conditions which cause dissociation of nAChRs from the cytoskeleton are the same that inhibit nAChR function and decrease agonist binding to cultured adrenal chromaffin cells. In conclusion, these studies support the hypothesis that there is an association of adrenal nAChRs with microtubules and suggest that the mechanism by which the microtubule drugs inhibit nAChR function is by inducing dissociation of nAChRs from the microtubular network. Furthermore, acetylated microtubules may be involved in localizing and anchoring of adrenal nAChRs on the cell membrane as has been implicated for muscle nAChRs (Jasmin et al., 1990). LIST OF REFERENCES Albuquerque, E.X., Tsai, M.C., Aronstram, R.S., Eldefrawi, A.T. and Eldefrawi, M.E. Sites of action of phencyclidine. II. Interaction with the ionic channel of the nicotinic receptor. Mol. Pharmacol., 18: 167-178, 1980. Allen, R.D., Weiss, D.G., Hayden, J.H., Brown, D.T., Fujiwake, H., Simpson, M. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an role of microtubules in cytoplasmic transport. J. Cell Biol., 100: 1736-1752, 1987. Allen, R.D. The Microtubule as Intracellular Engine. Sci. Am., 256: 42-49, 1987. Aunis, D., Guerold, B., Bader, M. F. and Cieselskitreska, J. Immuno-cytochemical and biochemical demonstration of contractile proteins in chromaffin cells in culture. Neuroscience, 5: 2261-2277, 1980. Avila, J. Microtubule dynamics. FASEB J., 4: 3284-3290, 1990. Baas, P.W. and Black, M.M. Individual microtubules in the axon consist of domains that differ in both composition and stability. J . Cell Biol., Ill: 495-509, 1990. Bader, M.F., Rousset, B., Bernier-Valentin, F. and Aunis, D. The adrenal paraneuron: tubulin organization. Can. J. Physiol. Pharmacol., 62: 502-511, 1984. Baker, P.F. and Rink, T.J. Catecholamine release from bovine adrenal medulla in response to maintained depolarization. J. Physiol. Lond., 253: 593-20, 1975. Banerjee, A.C. and Bhattacharyya, B. Colcemid and colchicine binding to tubulin. FEBS Lett., 99: 333-336, 1979. Banks, P. The adenosine triphosphatase activity of adrenal chromaffin granules. Biochem. J., 95: 490-496, 1965. 128 129 Barden, S., DePalma, R. and Gottesman, G. Crosslinking of proteins in acetylcholine receptor rich-membranes: Association between the beta-subunit and the 43Kd subsynaptic protein. Cell, 35: 687-692, 1983. Ben-Ze'ev, A., Duerr, A., Solomon, F. and Pennman, S. The outer boundary of the cytoskeleton: A lamina derived from plasma membrane proteins. Cell, 17: 859-865, 1979. Berra, H.S. Arce, C.A. Argarane, C.E. Posttranslational tyrosination-detyrosination of tubulin. Molec. Neurobiol., 2: 133-153, 1988. Bertrand, D., Ballivet, M. and Rungger, D. Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes. Proc. Natl. Acad. Sci. USA., 87: 1993-1997, 1990. Black, M.M. Aletta, J.M. amd Greene, L.A. Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth. J. Cell Biol., 103: 54 5- 557, 1986. Black, M.M. and Keyser, P. Acetylation of a-tubulin in cultured neurons and the induction of a-tubulin acetylation in PC12 cells by treatment with nerve growth. J.Neurosci., 7: 1833-1842, 1987. Black, M.M. and Baas, P.W. Individual microtubules in the axon consist of domains that differ in both composition and stability. J . Cell Biol., Ill: 495-509, 1990. Borisy, G.G. and Taylor, E.N. The mechanism of action of colchicine: Binding of 3H-colchicine to cellular proteins. J. Cell Biol., 34: 525-533, 1967. Boska, P. and Livett, B.G. Desensitization to nicotinic cholinergic agonists and potassium agents that stimulate catecholamine secretion in isolated adrenal chromaffin cells. J Neurochem 42: 607-617, 1984a. Boska, P. and Livett, B. G. Substance P protects against desensitization of the nicotinic response in isolated adrenal chromaffin cells. J. Neurochem., 42: 618-627, 1984b. Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinraann, S. and Patrick, J. Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc. Natl. Acad. Sci. USA., 84: 7763-7767, 1987. 130 Boulter, J. a3, a5, and B4: Three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J . Biol. Chem., 256: 4472-4482, 1990. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem., 72: 248-254, 1976. Brady, S.T., Lasek, R.L. and Allen, R.D. Fast axonal transport in extruded axoplasm from squid giant axon. Science, 218: 1129-1131, 1982. Brady, S.T., Tytell, M. and Lasek, R.J. Axonal tubulin and axonal microtubules: biochemical evidence for cold stability. J. Cell Biol., 99: 1716-1724, 1984. Brandt, B.L, Hagiwara, S., Kidokoro, Y. and Miyazaki, S. Action potentials in the rat chromaffin cell and effects of acetylcholine. J . Physiol. London., 263: 417-439, 1976. Brinkley, B.R., Cox, S.M. Pepper, D.A., Wiblie, L. Brenner, S.L. and Pardue, R.L. Tubulin assembly site and the organization of cytopalsmic microtubules in cultured mammalian cells. J . Cell Biol., 90: 554-562, 1981. Bryan, J.E. and Wilson, L. Are cytoplasmic microtubules heteropolymers? Proc. Natl. Acad. Sci., USA, 68: 1762-1766, 1971. Bunn, S.J. and Marley, P.D. Effects of angiotensin II on cultured bovine adrenal medullary cells. Neuropeptides 13: 121-132, 1989. Burgoyne, R.D. Control of exocytosis in adrenal chromaffin cells. Biochem. Biophys. Acta., 1071: 174-202, 1991. Burgoyne, R. D., Geisow, M. J., and Barron, Julie. Dissection of stages in exocytosis in adrenal chromaffin cell with use of trifluoperazine. Proc. Res. Soc. Lond., 216: 111-115, 1982. Burgoyne, R.D. Mechanisms of secretion from adrenal chromaffin cells. Biochim. Biophys. Acta 779: 201-216, 1984. Burgoyne, R.D. and Cheek, T.R. Reorganization of peripheral actin filaments as a prelude to exocytosis. Biosci. Rep., 7: 281-288, 1987. 131 Burgoyne, R.D., Cheek, T.R., Norman, K.M. Identification of a secretory granule-binding protein as caldesmon. Nature (Lond.), 319: 68-70, 1986. Carraway, K.L. and Carraway, C.A.C. Membrane-cytoskeleton interactions in animal cells. Biochim. Biophys. Acta, 988: 147-171, 1988. Chaliss, R.A.J., Jones, J.A., Owens, P.J. and Boarder, M.R. Changes in inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate mass accumulations in cultured adrenal chromaffin cells in response to bradykinin and histamine. J Neurochem 56: 1083-1086, 1991. Chiappinelli, V.A. kappa-Bungarotoxin: A probe for the neuronal nicotinic receptor in the avian ciliary ganglion. Brain Res., 227: 9-21, 1983. Chiappinelli, V.A., Lee, J.C. k-Bungarotoxin: self association of a neuronal nicotinic receptor probe. J. Biol. Chem., 260: 6182-6186, 1985. Clapman, D.E. and Neher, E. Substance P reduces acetylcholine induced currents in isolated bovine chromaffin cells. J. Physiol. (Lond.), 347: 255-277, 1984. Clarke, P.B., Pert, C.B. and Pert, A. Autoradiographic distribution of nicotinic receptors in rat brain. Brain Res., 323: 390- 395, 1984. Clarke, P.B., Schwartz, R.R., Paul, S.M., Pert, C.B. and Pert, A. Nicotinic binding in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine and lzSI-a- bungarotoxin. J . Neurosci., 5: 1307- 1315, 1985. Clineschmidt, B.V., Williams, M., Witoslawski, J.J., Bunting, P.R., Risley, E.A. and Totaro, J.A. Restoration of shock-suppressed behaviour by treatment with (+)-5-methyl- 10,ll-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine (MK- 801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties. Drug Dev. Res., 2: 147-163, 1982. Connolly, J.A. Role of the cytoskeleton in the formation, stabilization and removal of acetylcholine receptor clusters in cultured muscle cells. J . Cell Biol., 99: 148-154, 1984. Connolly, J.A. and Oldfin, B.V. Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells. J. Cell Biol., 100: 173-178, 1985. 132 Conti-Tronconi, B.M., Tang, F., Walgrave, S. and Gallagher, W. Nonequivalence of a-bungarotoxin binding sites in the native nicotinic receptor molecule. Biochemistry, 29: 1046- 1054, 1990. Cooke, P. H. and Poisner, A. M.: The role cytoskeleton in adrenal medullary secretion. In Methods and Achievements in Experimental Pathology, ed. by G. Jasmin and M. Cantin, vol. 9, pp. 137-146, S. Karger Publishers, Basel, 1979. Cooper, E., Couturier, S. and Ballivet, M. Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature, 350: 235-238, 1991. Corcoran, J. J. and Kirschner, N. Inhibition of calcium uptake, sodium uptake, and catecholamine secretion by methoxyverapamil (D600) in primary cultures of adrenal medulla cells. J . Neurochem., 40: 1106-1109, 1983. Couturier, S., Erkman, L., Valera, S., Rungger, D., Bertrand, S., Boulter, J., Ballivet, M. and Bertrand, D. a5, a3, and non-a3: Three clustered avian genes encoding neuronal nicotinic acetylcholine receptor-related subunits. J . Biol Chem., 265: 17560-17567, 1990. Cross, D., Dominguez, J., Maccioni, J.B. and Avila, J. MAP-1 and MAP-2 binding sites at the C-terminus of 6-tubulin, studies with synthetic tubulin peptides. Biochemistry, 30: 4362-4366, 1991. Das, M., and Lindstrom, J. The main immunogenic region of the nicotinic acetylcholine receptor. Interaction of monoclonal antibodies with synthetic peptides. Biochem. Biophys. Res. Comm., 165: 865-871, 1989. Deneris, E.S., Boulter, J., Swanson, L.W., Patrick, J. and Heinemann, S. 63: A new member of nicotinic acetylcholine receptor gene family is expressed in brain. J. Biol. Chem., 264: 6268-6272, 1989. Deneris, E.S., Connolly, J., Rogers, S.W. and Duvoisin, R. Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors. Trends Pharmacol., 12: 34-40, 1991. Devreotes, P.N. and Fambrough, D.M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J . Cell Biol., 65: 335-358, 1975. 133 Diaz-Nido, J., Serrano, L., Hernandez, M. and Avila, J. Phosphorylation of microtubule protiens in rat brain at different developmental stages: comparison with that found in neuronal cultures. J. Neurochem., 54: 211-221, 1990. Douglas, W. W. and Rubin, R. P. Stimulant action of barium on the adrenal medulla. Nature (Lond.), 203: 305-307, 1964. Douglas, W. W. and Poisner, A. M. Preferential release of adrenaline from the adrenal medulla by muscarine and pilocarpine. Nature, Lond., 208: 1102-1103, 1965. Douglas, W. W. and Sorimachi, M. Colchicine inhibits adrenal medullary secretion evoked by acetylcholine without affecting that evoked by potassium. Br. J. Pharmacol., 45: 129-132, 1972. Douglas, W.W. Secretomotor control of adrenal medullary secretion: Synaptc membrane and ionic events in stimulus- secretion coupling. In: Handbook of Physiology. Endocrinology. Adrenal Gland. Washington D.C.: Am Physiol Soc, Sect. 7, Vol. 6, Chapter 26, pp 367-388, 1975. Douglas, W. W., Kanno, T. and Sampson, S. R. Effects of acetylcholine and other medullary secretagogues and antogonists on the membrane potential of the adrenal chromaffin cells: an analysis employing techniques of tissue culture. J. Physiol. London, 188: 107-120, 1967. Duanmy, Chi, Shahrik, L.K., Ho, H.H. and Hamel, E. Tubulin- dependent hydrolysis of guanosine triphosphate as a screening test to identify new antitubulin compounds with potential as antimitotic agents: Application to carbamates of aromatic amines. Cancer Res., 49: 1344-1348, 1989. Dustin, P. Microtubules. Springer-Verlag, N.Y., N.Y., 1984. Duvoisin, R.M., Deneris, E.S., Patrick, J. and Heinemann, S. The functional diversity of the neuronal nicotinic receptors is increased by a novel subunit: 64. Neuron, 3: 487-496, 1989. Eldefrawi, A.T. and Eldefrawi, M.E., Albuquerque, E.X., Oliviera, A.C., Mansour, N., Adler, M., Daly, J.W., Brown, G.B., Burgermeister, W. and Witkop, B. Perhydrohistrionicotoxin: a potential ligand for the ion conductance modulator of the acetylcholine receptor. Proc. Natl. Acad. Sci. USA., 74: 2172-2176, 1977. 134 Erickson, H.P. and Voter, W. Nucleation of microtubule assembly. Ann. N.Y. Acad. Sci., 466: 552-565, 1986. Falconer, M.M., Vielkind, U. and Brown, D.L. Establishment of a stable, acetylated microtubule bundle during neuronal commitment. Cell Motil. Cyt., 12: 169-180, 1989. Fambrough, D.M. Control of acetylcholine receptors in skeletal muscle. Physiol. Rev., 59: 165-226, 1979. Feit, H., Dutton, G., Barondes, S. and Shelanski, M. Microtubule proteins: identification in and transport to nerve endings. J. Cell Biol., 57: 138-147, 1971. Flavin, M. and Slaughter, C. Microtubule assembly and function in Chlamydomonas. Inhibition of growth and flagellar regeneration by antitubulins and otherdrugs and isolation of resistant mutants. J. Biol. Chem., 118: 59-69, 1974. Fornasari, D., Chini, B., Tarroni, P. and Clementi, F. Molecular cloning of human neuronal nicotinic receptor a3-subunit. Neurosci. Lett., Ill: 351-356, 1990. Freedman, R.B. In: Membrane strucuture, J.B. Finean and R.H. Michell, editors, pp 161-214, Elsevier, Amsterdam, 1981. Froehner, S.C. The role of the postsynaptic cytoskeleton in AChR organization. Trends Neurosci., 9: 37-41, 1986. Froehner, S.C., Luetje, C.W., Scotland, P.B. and Patrick, J. The postsynaptic 4 3K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron, 5: 403- 410, 1990. Froehner, S.C., Gulbrandsen, V., Hyman, C., Jeng, A.Y., Neubig, R.R. and Cohen, J.B. Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membrane. Proc. Natl. Acad. Sci. USA, 78: 5230-52 34, 1981. Fujiwara, K. and Tilney, L.G. Substructural analysis of the microtubule and its polymorphic forms. Ann. N.Y. Acad. Sci., 253: 27-50, 1975. Fulton, A., Prives, J., Farmer, S. and Penmann, S. Development reorganization of the skeletal framework and its surface lamina in fusing muscle cells. J . Cell Biol., 91: 103-112, 1981. 135 Galzi, J., Revah, F., Bessis, A. and Changeux, J.-P. Functional architecture of the nicotinic acetylcholine receptor: from electric organ to brain. Annu. Rev. Pharmacol., 31: 37-72, 1991. Gard, D.L. and Kirschner, M.A. Polymer-Dependent Increase in Phosphorylation of B-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J . Cell Biol., 100: 764- 774, 1985. Geuens, G.M.A., Nuydens, R.M., Willebrords, R.E., Van deVeire, R.M.L., Goossensl, F., Dragonetti, C.H., Mareel, M.M.K. and DeBrabander, M.J. Effects of tubulozole on the microtubule system of cells in culture and in vitro. Cancer Res., 45: 733-742, 1985. Gillespie, E., Levine, R.J. and Malawista, S.E. Histamine release from rat peritoneal mast cells: inhibition by colchicine and potentiation by deuterium oxide. J. Pharmacol. Exp. Ther., 164: 158-165, 1968. Gozes, I. and Sweadner, K.J. Multiple tubulin forms are expressed by a single neuron. Nature (Lond.), 294: 477-480, 1981. Gunderson, G.G., Kalnoski, M.H. and Bulinski, J.C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell, 38: 779-789, 1984. Gunderson, G.G., Khawaja, S. and Bulinski, J.C. Postpolymerization detyrosination of a-tubulin: a mechanism for subcellular differentiation of microtubules. J. Cell Biol., 105: 251-264, 1987. Halvorsen, S.W. and Berg, D.K. Identification of a nicotinic acetylcholine receptor on neurons using an a-neurotoxin that blocks receptor function. J. Neurosci., 6: 3405-3412, 1986. Halvorsen, S.W. and Berg, D.K. Affinity labeling of neuronal acetylcholine receptors with an a-neurotoxin that blocks receptor function. J. Neurosci., 7: 2547-2555, 1987. Hamel, E., del Campo, A.A., Lustbader, J. and Lin, C.M. Modulation of tubulin-nucleotide interactions by microtubule associated proteins. Biochemistry, 22: 1271-1279, 1983. 136 Hamano, S. H., Morita, K., Azuma, M., Oka, M., and Teraoka, K. Pharmacological study on Ba+2 -stimulated catecholamine secretion from cultured bovine adrenal chromaffin cells: Possible relation of Ba+2 action to Ca+2 -activated secretory mechanism. Japan. J. Pharmacol., 55: 43-50, 1991. Harlow, E. and Lane, D. Growing Hybridomas In: Antibodies a Laboratory Manual, chap. 7, pgs. 245-282, Cold Spring Harbor Laboratory, N.Y., 1988. Harlow, E. and Lane, D. Storing and purifying antibodies In: Antibodies a Laboratory Manual, chap. 8, pgs. 283-318, Cold Spring Harbor Laboratory, N.Y., 1988. Hayden, J. and Allen, R.D. Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule. J. Cell Biol., 98: 1785-1793, 1984. Heinemann, S., Merlie, J., and Lindstrom, J. Modulation of acetylcholine receptor in rat diaphragm by anti-receptor sera. Nature, 274: 65-68, 1978. Henis, Y.I. Lateral mobility measurement of cell surface components: applications for molecular pharmacology. Trends Pharmacol., 10: 95-98, 1989. Higgins, L.S. and Berg, D.K. Immunological identification of a nicotinic acetylcholine receptor on bovine chromaffin cells. J . Neurosci., 7: 1792-1798, 1987. Higgins, L.S. and Berg, D.K. Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells. J . Cell Biol., 107: 1147- 1156, 1988a. Higgins, L.S. and Berg, D.K. A desensitized form of neuronal acetylcholine receptor detected by 3H-nicotine binding on bovine adrenal chromaffin cells. J. Neurosci., 8: 1436- 1446, 1988b. Higuchi, H., Takeyasu, K., Uchida, S. and Yoshida, H. Receptor activated and energy-dependent decrease of muscarinic cholinergic receptors in guinea pig vas deferens. Eur. J. Pharmacol., 75: 305-311, 1981. Himes, R.H., Kersey, R.N., Heller-Bettinger, I.J. and Samson, F.E. Action of the vinca alkaloids, vincristine, vinblastine and desacetylvinblastine amide on microtubules in vitro. Cancer Res., 36: 3798-3800, 1977. 137 Hoebeke, J. and VanNijen, G. Qualitative turbidity assay for potency evaluation of colchicine-like drugs. Life Sci., 17: 591-601, 1975. Holz, R. W., Senter, R. A. and Frye, R. A. Relationship between Ca2+ uptake and catecholamine secretion in primary dissociated cultures of adrenal medulla. J. Neurochem., 39: 635-646, 1982. Horwitz, A., Duggan, K., Buck, C., Beckerle, M.C. and Burridge, K. Interaction of plasma membrane fibronectin receptor with talin-A transmembrane linkage. Nature, 32 0: 531-533, 1986. Hucho, F. The nicotinic acetylcholine receptor and its ion channel. J. Biochem., 158: 211-226, 1986. Hunt, R.C., Dewey, A. and Davis, A.A. Transferrin receptors on the surfaces of retinal pigment epithelial cells are associated with the cytoskeleton. J. Cell Sci., 92: 655- 666, 1989. Ito, S., Mochizuki-Oda, N., Hori, K., Ozaki, K., Miyakawa, A. and Negishi, M. Characterization of prostaglandin E- induced Ca+2 mobilization in single bovine adrenal chromaffin cells by digital image microscopy. J Neurochem 56: 531-540, 1991. Jacob, M.H., Berg, D.K. and Lindstrom, J.M. Shared antigenic determinant between Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons. Proc. Natl. Acad. Sci. USA., 81: 3223-3227, 1984. Jasmin, B.J., Changeux, J.-P. and Cartaud, J. Compartmentalization of cold-stable and acetylated microtubules in the subsynaptic domain of chick skeletal muscle fibre. Nature (Lond.), 344: 673-675, 1990. Jordan, M.A. and Farsell, K.W. Differential radiolabeling of opposite microtubules ends: methodology, equilibrium exchange-flux analysis and drug poisoning. Ann. Biochem., 130: 241-248, 1983. Kelleher, J.K. Tubulin binding affinities of podophyllotoxin and colchicine analogues. Molec. Pharmacol., 13: 232-241, 1977. Kidokoro, Y., Miyazaki, S. and Osawa, S. Acetylcholine- induced membrane depolarization and potential fluctuations in the rat adrenal chromaffin cells. J. Physiol. (Lond.), 324: 203-220, 1982. 138 Kilpatrick, D.L., Slepetis, R. and Kirschner, N. Inhibition of catecholamine secretion from adrenal medulla cells by neurotoxins and cholinergic antagonist. J. Neurochem., 37: 125-131, 1981. Kilpatrick, D. L., Slepetis, R. J., Concoran, J. J. , and Kirschner, N. Calcium uptake and catecholamine secretion by cultured bovine adrenal medulla cells. J. Neurochem., 38: 427-435, 1982. Kilpatrick, D. L., Slepetis, R. and Kirschner, N. Ion channels and membrane potential in stimulus-secretion coupling in adrenal medulla cells. J . Neurochem., 36: 1245- 1255, 1981. Kondo, T.H., Wolosewick, J.J. and Pappas, G.D. The microtrabecular lattice of the adrenal medulla revealed by polyethylene glycol embedding and stereo electron microscopy. J. Neurosci., 2: 57-65, 1982. L'Hernault, S.W. and Rosenbuum, V.L. Chlamidomonas tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine. Biochemistry, 24: 473-478, 1985. Lacy, P. E., Howell, S. L. and Young, D. A. New hypothesis of insulin secretion. Nature (London), 219: 1177-1179, 1968. LaRochelle, W.J., Witzemann, V., Fiedler, W. and Froehner, S.C. Developmental expression of the 4 3K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes. J. Neurosci., 1986 Levy, D.A. and Carlton, J.A. Influence of temperature on the inhibition by colchicine of allergic histamine release. Proc. Soc. Exp. Biol. Med., 130: 1333-1336, 1969. Lewis, S.A., Inanov, I.E., Lee, G.H. and Cowan, N.J. Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule- associated proteins MAP2 and tau. Nature, 342: 498-505, 1989. Lindstrom, J., Whiting, P., Schoepfer, R., Luther, M. and Das, M. Structure of nicotinic acetylcholine receptors from muscle and neurons. Computer assisted modeling of receptor- ligand interactions: theoretical aspects and applications to drug design. Prog. Clin. Biol. Res., 289: 245-266, 1989. 139 Lindstrom, J., Einarson, B. and Tzartos, S. Production and assay of antibodies to acetylcholine receptors. Meth. Enzymol., 74: 432-460, 1981. Lindstrom, J., Schoepfer, R., Whiting, P. Molecular studies of the neuronal nicotinic acetylcholine receptor family. Molec. Neurobiol., 1: 218-3 37, 1987. Lippiello, P.M. and Fernandes, K.G. The binding of L- [3H]nicotine to a single class of high affinity sites in rat brain membranes. Molec. Pharmacol., 29: 448-454, 1986. Lippiello, P.M., Sears, S.B. and Fernandes, K.G. Kinetics and Mechanism of L-[3H]nicotine binding to putative high affinity receptor sites in rat brain. Mol. Pharmacol., 31: 392-400, 1987. Lipscombe, D. and Rang, H.P. Nicotinic receptors of frog ganglia resemble pharmacologically those of skeletal muscle. J . Neurosci., 8: 3258-3265, 1988. Lipton, S., Aizeman, E. and Loring, R.H. Neuronal nicotinic acetylcholine reponses in mammalian retinal ganglion cells. Pfliigers Arch., 410: 37-4 3, 1987. Livett, B. G., Boksa, P., Dean, D. M. , Mizobe, F. and Lindenbaum, M. H. Use of isolated chromaffin cells to study basic release mechanisms. J. Autonomic Nerv. Syst., 7: 59- 86, 1983. Livett, B.G. Adrenal medullary chromaffin cells in vitro. Physiol. Rev., 64: 1103-1161, 1984. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. Protein measurement with the folin phenol reagent. J . Biol. Chem. 193: 65-275, 1951. Luduena, R., Wilson, L. and Shoota, E.M. Crossbinding of tubulin: evidence for the heterodimer model. J. Cell Biol., 63: 202-210, 1964. Luduena, R. F., Fellous, A., Francon, J., Nunez, J. and McManus, L. Effect of tau on the vinblastine-induced aggregation of tubulin. J . Cell Biol. 89:680-683, 1981. Luetje, C.W., Wada, K., Rogers, S., Abramson, S.N., Tsuji, K., Heinemann, S. and Patrick, J. Neurotoxins distinguish between different neuronal nicotinic acetylcholine receptors. J . Neurochem., 55: 632-640, 1990. 140 Luetje, C.W. and Patrick, J. Both a- and fi-subunits contribute to the agonist sensitivity of neuronal nicotinic acetylcholine receptors. J. Neurosci., 11: 837-845, 1991. Manfredi, J.J. and Horwitz, S.B. Taxol: An antimitotic agent with a new mechanism of action. Pharmacol. Ther., 25: 83-125, 1984. Marks, M.J., Stitzel, J.A., Romano, E., Wehner, J.M. and Collins, A.C. Nicotinic binding sites in rat and mouse brain: comparison of acetylcholine, nicotine and a- bungarotoxin. Mol. Pharmacol., 30: 427-43&, 1986. Marley, P.D. Desensitization of the nicotinic secretory response of adrenal chromaffin cells. Trends Pharm Sci., 9: 102-106, 1988. Maruta, H., Green, K. and Rosenbaum, J.L. The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules. J. Cell Biol., 103: 571-579, 1986. Marute, H. Green, K. and Rosenbaum, J.L. The acetylation of alpha-tubulin and its relationship to the assembly and dessembly of microtubules. J . Cell Biol., 103: 571-580, 1986. Matus, A. Microtubule-associated proteins: their potential role in determining neuronal morphology. Annu. Rev. Neurosci., 11: 29-44, 1988. McKay, D.B. and Schneider, A.S. Selective inhibition of cholinergic receptor-mediated 45Ca+2 uptake and catecholamine seretion from adrenal chromaffin cells by taxol and vinblastine. J . Pharmacol. Exp. Ther., 231: 102- 108, 1984. McKay, D.B., Aronstam, R.S. and Schneider, A.S. Interactions of microtubule-active agents with nicotinic acetylcholine receptors. Relationship to their inhibition of catecholamine secretion by adrenal chromaffin cells. Molec. Pharmacol., 28: 10-16, 1985. McKay, D.B., Cobianchi, M.J. and Schneider, A.S. Comparison of the effects of colchicine and beta-lumicolchicine on cultured adrenal chromaffin cells: lack of evidence for an action of colchicine on receptor-associated microtubule. Pharmacology, 35: 155-162, 1987. McKay, D.B. Structure-activity study on the actions of taxol and related taxanes on primary cultured adrenal medullary cells. J . Pharmacol. Exp. Ther., 248: 1302-1307, 1988. 141 McKay, D.B. [3H]Noradrenaline accumulation in cultured bovine adrenal medullary cells: modulation of accumulation by nicotine. Naunyn-Schmiedeberg1s Arch. Pharmacol., 340: 610-616, 1989. McKay, D.B. and Trent-Sanchez, P. Effect of noncompetitive nicotinic receptor blockers on catecholamine release from cultured adrenal chromaffin cells. Pharmacology, 825: 1-7, 1990. Melmed, R.N., Karanian, P.J. and Berlin, R.D. Control of cell volume -in the J774 macrophage by microtubule disassembly and cyclic AMP. J. Cell Biol., 90: 761-768, 1981. Merlie, J.P., Heinemann, S. and Lindstrom, J. Acetylcholine receptor degradation in adult rat diaphragm in organ cultures and the effect of anti-acetylcholine receptor antibodies. J. Biol. Chem., 254: 6320-6327, 1979. Mitchison, T. and Kirschner, M. Dynamic instability of microtubule growth. Nature (Lond.), 312: 237-242, 1984. Morley, B.J. and Kemp, G.E. Characterization of a putative nicotinic acetylcholine receptor in mammalian brain. Brain Res. Rev., 3: 81-104, 1981. Mraz, P. Effects of some drugs upon cytoskeletal microtubules as revealed by immunofluorescence technique with monoclonal antitubulin antibodies. Z. Mikrosk.-Anat. Forsch., 99: 911-919, 1985. Nicolson, G. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over cell surface components. Biochem. Biophys. Acta., 457: 57-108, 1976. O'Sullivan, A.J. and Burgoyne, R.D. A comparison of bradykinin, angiotensin II and muscarinic stimulation of cultured adrenal chromaffin cells. Biosci. Rep., 9: 243- 252, 1989. Olmsted, J.B. Microtubule associated proteins. Ann. Rev. Cell Biol., 2: 421-457, 1987. Orci, L., Gabbay, K.H. and Malaisse, W.J. Pancreatic beta cell web: Its possible role in insulin secretion. Science, 175: 1128-1130, 1972. Oswald, R.E. and Freeman, J.A. Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors. Neuroscience, 6: 1-14, 1981. 142 Owellen, R.J., Owens, A.H. and Donigian, D.W. The binding of vincristine, vinblastine and colchicine to tubulin. Biochem. Biophys. Res. Comm., 47: 685-691, 1972. Papke, R.L., Boulter, J., Patrick, J. and Heinemann, S. Single channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neuron, 3: 589-596, 1989. Patrick, J. and Stallcup, W.B. Immunological distiction between acetycholine receptor and the alpha bungarotoxin binding component on sympathetic neurons. Proc. Natl. Acad. Sci., 74: 4689-4692, 1977. Penningroth, S.M. and Kirschner, M.W. Nucleotide binding and phosphrylation in microtubule assembly in vitro. J. Molec. Biol., 115: 643-673, 1977. Peters, A., Palay, S.L. and Webster, H. The fine structure of the nervous system. W.B. Saunders Co., NY, NY, 1976. Phillips, W.D., Kopta, C., Blount, P., Gardnner, P.D., Steinbach, J.H. and Merlie, J.P. Acetylcholine receptor rich membrane domains organized in fibroblasts by recombinant 4 3 kilodalton protein. Science, 251: 568-570, 1991. Piperno, G., LeDizet, M., Chang, X. Microtubules containing acetylated a-tubulin in mammalian cells in culture. J. Cell Biol., 104: 289-302, 1987. Piperno, G. and Fuller, M.T. Monoclonal antibodies specific for an acetylated from of a-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J . Cell Biol., 101: 2085-2094, 1985. Plevin, R. and Boarder, M.J. Stimulation of formation of inositol phosphates in primary cultures of bovine adrenal chromaffin cells by angiotensin II, histamine, bradykinin and carbachol. J. Neurochem. 51: 634-641, 1988. Poisner, A. M. and Bernstein, J. A possible role of microtubules in catecholamine release from the adrenal medulla. Effect of colchicine, vinca alkaloids and deuterium oxide. J. Pharmacol. Exp. Ther., 177: 102- 108, 1971. Poisner, A.M. and Trifaro, J.-M. The role of adenosine triphosphate and adenosine triphosphatase in the release of catecholamines from the adrenal medulla. I. ATP-evoked release of catecholamines, ATP and protein from isolated chromaffin granules. Mol. Pharmacol., 3: 561-571, 1967. 143 Prives, J., Fulton, A.B., Penman, S., Daniel, M.P. and Christian, C.N. Interaction of the cytoskeletal framework with acetylcholine receptor on the surface of embryonic muscle cells in culture. J . Cell Biol., 92: 231-236, 1982. Ralston, S., Sarin, V., Thanh, H., Rivier, J., Fox, J.L. and Lindstrom, J. Synthetic peptides used to locate the alpha- bungarotoxin binding site and immunogenic regions on alpha subunits of the nicotinic acetylcholine receptor. Biochemistry, 26: 3261-3266, 1987. Ramoa, A.S., Alkondon, M., Aracave, Y., Ivons, J. , Lunt, G.G., Des Heande, S.S., Wonnacott, S., Aronstam, R.S. and Albuquerque, E.X. The anticonvulsant MK-801 interacts with the peripheral and central nicotinic acetylcholine receptor ion channels. J. Pharmacol. Exp. Ther., 254: 79-82, 1990. Rapier, C., Wonnacott, S., Lunt, G.G. and Albuquerque, E.X. The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system. FEBS Lett., 212: 292-296, 1987. Ratnam, M., Sargent, P.B., Sarin, V. , Fox, J.L., LeNguyen, D., Rivier, J., Criado, M. and Lindstrom, J. Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochemistry, 25: 2621-2632, 1986a. Ray, K., Bhattacharyya, B. and Biswas, B.B. Role of B-ring of colchicine in its binding to tubulin. J . Biol. Chem., 256: 6241-6244, 1981. Romano, C. and Goldstein, A. Stereospecific nicotine receptors on rat brain membranes. Science, 210: 647-649, 1980. Sahenk, Z. and Brady, S.T. Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules. Cell Motil. Cytoskel., 8: 155-164, 1987. Sakmann, B., Bormann, J. and Hamill, O. Ion transport by single receptor channels. Cold Spring Harbor Symposia on quantitative biology 48: 247-257, 1983. Sasakawa, N., Ishii, K. and Kato, R. Calcium-independent desensitization of rises in intracellular free Ca 2 concentration and catecholamine release in cultured adrenal chromaffin cells. Biochem. Biophys. Res. Comm., 133: 147- 153, 1985. 144 Sawrok, E., Schloss, P., Betz, H. and Schmitt, B. Heterogeneity of Drosophila nicotinic acetylcholine receptors. SAD: a novel developmentally regulated a-subunit. EMBOJ., 9: 2671-2677, 1990. Schnapp, B.J., Vale, R.D., Sheetz, M.P. and Reese, T.S. Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell, 40: 455-462, 1985. Schoepfer, R., Conroy, W.G., Whiting, P., Bore, M. and Lindstrom, J. a-Bungarotoxin binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron, 5: 35-48, 1990. Schoepfer, R., Whiting, P., Luther, M., Keyser, K. , Karten, H. and Lindstrom, J. Structure of muscle and neuronal nicotinic acetylcholine receptors. Molecular biology of neuroreceptors and ion channels, editor: A. Maelilcke, Springer-Verlag, Berlin Heidelberg, 1989. Schroer, T.A. and Sheetz, M.P. Function of microtubule-based motors. Ann. Rev. Physiol., 53: 629-652, 1991. Schultz, E. and Brady, S.T. Axonal tubulin and microtubules: morphological evidence for stable regions on axonal Microtubules. Cell Motil. Cyt., 8: 155-164, 1987. Schulze, E. and Kirschner, M. Dynamic and stable populations of microtubules in cell. J. Cell Biol., 104: 277-288, 1987. Sealock, R., Wray, B.E. and Froehner, S.C. Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies. J. Cell Biol., 98: 2239-2244, 1984. Smith, M. A., Stollberg, J., Lindstrom, J. M. , and Berg, D. K. Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor. J. Neuroscience, 5: 2726-2731, 1985. Stendahl, O. I. and Stossel, T. P. Actin-binding protein amplifies actinomycin contraction, and gelsolin confers calcium control on the direction of contraction. Biochem. Biophys. Res. Commun., 92: 675-681, 1980. Stya, M. and Axelrod, D. Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubules: a correlation. J. Cell Biol., 97: 48-51, 1983a. 145 Stya, M. and Axelrod, D. Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes. Proc. Natl. Acad. Sci. USA., 80: 449-453, 1983b. Trifaro, J.-M. Common mechanisms of hormone secretion. Annu. Rev. Pharmacol. Toxicol., 17: 27-47, 1977. Trifaro, J.-M. Cellular and molecular mechanisms in hormone and neurotransmitter secretion. Can. J. Physiol. Pharmacol. 68: 1-16, 1989. Trifaro, J.-M., Collier, B., Lastowecka, A. and Stern, D. Inhibition by colchicine and vinblastine of acetylcholine- induced catecholalmine release from the adrenal gland: an acetylcholine action not an effect upon microtubules. Molec. Pharmacol., 8: 264-267, 1972. Trifaro, J. M., Kenigsberg, R. L., Cote, A., Lee, R. W. H. , and Hikita, T. Adrenal paraneurone contractile proteins and stimulus-secretion coupling. Can. J. Physiol. Pharmacol., 62: 493-501, 1983. Trifaro, J.-M. and Lee, R. W. H. Morphological characteristics and stimulus-secretion coupling in bovine adrenal chromaffin cell cultures. Neuroscience, 5:1533- 1546, 1980. Trifaro, J.-M., Ulpian, C. and Preiksaitis, H. Anti-myosin stains chromaffin cells. Experientia, 34: 1568-1571, 1978. Tzartos, S., Rand, D., Einarson, B., and Lindstrom, J. Mapping of surface structures on electrophorus acetylcholine receptor using monoclonal antibodies. J. Biol. Chem., 256: 8635-8645, 1981. Tzartos, S., Kokla, A., Walgrave, S. L., and Conti-Tronconi, B. The main immunogenic region of human muscle acetylcholine receptor is localized within residues 63-80 of the a-subunit. Proc. Natl. Acad. Sci., 85: 2899-2903, 1988. Unsiker, K. B., Rifferert, B. and Ziegler, W. Effects of cell culture conditions, nerve growth factor, dexamethazone, and cyclic AMP on adrenal chromaffin cells in vitro. Adv. Biochem. Psychopharmacol., 25: 51-59, 1980. Vale, R. Intracellular transport using Microtubule-based motors. Annu. Rev. Cell Biol., 3: 347-378, 1987. 146 Vale, R.D. and Shooter, E.M. Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors on PC12 cells by wheat germ agglutinin. J. Cell Biol., 94: 710-717, 1982. Vallee, R.B., Bloom, G.S. and Theurkauf, W.E. Microtubule Associated Proteins: Subunites of the Cytomerines. J. Cell Biol., 99: 385-445, 1987. Van Ginckel, R.., DeBrabander, M., Vanherck, W. and Heeres, J. The effects of tubulozole, a new synthetic microtubule inhibitor on experimental neoplasm. -Fur. J. Cancer Clin. Oncol., 22: 99-105, 1984. Viveros, 0. H., Aggueros, L. and Kirschner, N. Release of catecholamines and dopamine 0-oxidase from the adrenal medulla. Life Sci., 7: 609-618, 1968. Viveros, O. H. and Wilson, S. P. The adrenal chromaffin cell as a model to study the co-secretion of enkephalins and catecholamines. J. Auto. Nerv. Syst., 7: 41-58, 1983. Wada, K., Ballivet, M. , Boulter, J., Connolly, J. , Wada, E., Deneris, E.S., Swanson,L.W., Heinemann, S. and Patrick, J. Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor. Science, 240: 330- 334, 1988. Weisenberg, R.C. and Deery, W.J. Role of nucleotide hydrolysis is microtubule assembly. Nature (Lond.), 263: 792-793, 1976. Wennogle, L.P. and Changeux, J.-P. Transmembrane orientation of proteins present in acetylcholine receptor-rich membranes from Torpedo marmorata studied by selective proteolysis. Eur. J. Biochem., 106: 381-393, 1980. Whiting, P. and Lindstrom, J. Purification and characterization of a nicotinic acetylcholine receptor from chick brain. Biochemistry, 25: 2082-2093, 1986. Whiting, P. and Lindstrom, J. Purification and characterization of a nicotinic acetylcholine receptor from rat brain. Proc. Natl. Acad. Sci. USA., 84: 595-599, 1987a Whiting, P. and Lindstrom, J. Affinity labelling of neuronal acetylcholine-receptors localizes acetylcholine-binding sites to their 0-subunits. FEBS. Lett., 213: 55-60, 1987b. 147 Whiting, P., Liu, R., Morley, B.J. and Lindstrom, J. Structurally different neuronal nicotinic acetylcholine receptor subtypes purified and characterized using monoclonal antibodies. J. Neurosci., 7: 4005-4016, 1987. Wilson, L. Microtubules as drug receptors: Pharmacological properties of microtubule protein. Ann. N.Y. Acad. Sci., 253: 213-231, 1975. Wilson, S.P. Vasoactive intestinal peptide elevates cyclic AMP levels and potentiates secretion in bovine adrenal chromaffin cells. Neuropeptides 11: 17-21, 1988. Wilson, L., Banburg, J.R., Mizel, S.B., Grisham, L.M. and Creswell, K.M. Interaction of drugs with microtubule protein. Fed. Proc., 33: 158-166, 1978. Young, S. H., and Poo, M. M. Topographical rearrangement of acetylcholine receptors alters channel kinetics. Nature, 304: 161-163, 1983. Zippel, R., Morello, L., Brambilla, R., Comeglia, P.M., Alberghina, L. and Sturani, E. Inhibition of phophotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase. Eur. J. Cell Biol., 50: 428-434, 1989.